Systems and methods for monitoring health and delivering drugs transdermally

ABSTRACT

The present invention pertains to a system and method for transdermal sampling, comprising: at least one sampler for retrieving and transferring at least one analyte obtained transdermally from the skin of a subject; at least one detector system for identifying and quantifying said at least one analyte; and at least one logic module for (i) receiving and storing input data from said at least one detector, (ii) relating the input data to other data obtained from the subject, (iii) displaying output information, (iv) transmitting the output information to another system, and (v) controlling the operation of said at least one sampler and at least one detector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and incorporates by reference in itsentirety provisional application Ser. No. 60/208,327, filed Jun. 1, 2000entitled “TRANSDERMAL HEALTH MONITORING AND DRUG DELIVERY SYSTEM”.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to portable biomedicalmonitoring. More specifically, this invention relates to non-invasiveand minimally invasive molecular monitoring, and optionally theimplementation of protective feedback measures and remote monitoringthrough telemetry.

2. Description of the Related Art

Non-invasive transdermal sampling of body fluids has long been a goal inmedical research. The notion that valuable diagnostic informationcomprising the concentrations of key analytes within the bloodstreamcould be obtained without breaching the skin has spurred many lines ofresearch. With such technology, long-term convenient health monitoringand screening without needles or outpatient care would become a reality:diabetics could monitor blood glucose without drawing blood; markers formicrobial, fungal or viral infections could be monitored; andenvironmental exposure to toxins could be assessed non-invasively.

Biomarkers have been utilized effectively to detect, measure, and assessexposure levels to environmental chemicals deemed hazardous and toxic tohuman life. The sensitivity of biomarkers allows them to act as earlywarning indicators to subtle alterations in the environment. Theirspecificity can be used to establish the nature of the imposing chemicalagent, determine exposure level and define a suitable course of action.Environmentally induced diseases affect everyone to one degree oranother, however individual susceptibilities can predispose the degreeof toxic reaction of one group over another. It is worthwhile notingthat in 1996, there were 86,912 cases of pesticide exposures reported toAmerican Association of Poison Centers, of which 26 were fatalities. Inparticular, individuals in their developmental stages, ranging from theembryonic phase to adolescence, are particularly susceptible to suchenvironmental stresses since key body functions have not matured to alevel where they can tolerate, process and handle such exposures. Theuse of biomarkers for determination of children's environmental healthwill allow for the early detection of toxins, prevention of impairmentin their physical condition, and determine a course of treatment forchildren who have; been exposed to a toxic environment.

Especially important in the field of pediatrics is the use of healthevaluation tools that are minimally intrusive.

Many transdermal sampling techniques have been reported, but all to datesuffer from one or more serious drawbacks. Conventional techniques havedisadvantages of being grossly invasive (and potentially injurious) andsweat or interstitial fluid dependent, except for the: passive,non-sweat dependent transdermal analyte collection and detectiontechniques.

One approach to transdermal sampling has employed the collection ofsweat. For example, M. Philips and M. H. McAloon. Alcohol Clin. Exp.Res. 4 391 (1980) disclose an adsorbent patch which is asalt-impregnated, cellulose pad under an occlusive, adhesive cover.However, such a method of transdermal sampling is dependent upon thesweat rate, requires sweat extraction by centrifugation, and calls forexternal chemical analysis. S. Balabanova and E. Schneider. Beitr.Gerichtl. Med 48, 45 (1990) disclose Pilocarpine-induced sweatsecretion, but the system requires Iontophoresis-induced infusion ofpilocarpine and analyte dilution. U.S. Pat. No. 5,203,327, issued toSchoendorfer et al., discloses an absorbant pad under a watervapor-permeable, occlusive, adhesive cover, but the system is sweat ratedependent and requires chemical extraction and external chemicalanalysis. F. P. Smith and D. A. Kidwell, Forensic Sci. Int. 83, 179(1996) discloses a cotton sweat wipe, but this system is sweatvolume-dependent and requires extraction and external chemical analysis.G. L. Henderson and B. K. Wilson,. Res. Commun. Chem. Pathol. Pharmacol.5, 1 (1973) discloses the collection of liquid sweat following exercise,but the system requires vigorous exercise, is sweat volume-dependent,and requires extraction and external chemical analysis.

C. C. Peck, D. P. Conner, et al. Skin Pharmacol 1, 14 (1988) discloses agel with an analyte binding reservoir under an occlusive adhesive cover.However, this reference requires extraction and external chemicalanalysis.

U.S. Pat. No. 4,909,256, issued to Peck discloses a dry bindingreservoir under an occlusive adhesive cover. However, this referencerequires extraction and external chemical analysis.

U.S. Pat. No. 4,821,733, issued to Peck discloses a collection anddetection system under an occlusive adhesive cover. However, thisreference requires highly sensitive detection components.

U.S. Pat. No. 4,775,361, issued to Jacques discloses enhanced migrationof analyte to a skin surface. However, this reference requiresintroduction of light energy into the body.

U.S. Pat. No. 5,362,307, issued to Guy discloses iontophoretic enhancedanalyte collection across skin. However, this reference requires theintroduction of electrical energy into the body.

U.S. Pat. No. 5,722,397, issued to Eppstein discloses ultrasoundenhanced analyte collection across skin. However, this referencerequires the introduction of sonic energy and chemicals into the body.

U.S. Pat. No. 5,885,211, issued to Eppstein, discloses microporeformation using heated water vapor, physical lancet, sonic energy, highpressure jet of fluid, or electricity. However, this reference requirespuncture of the skin using heat, sonic, or electrical energy, physicalor hydraulic force.

The website www.spectrx.com discloses the application of vacuum tolaser-induced dermal micropores for harvesting of interstitial fluid.However, this reference requires introduction of sonic energy into body,as well as physical energy to harvest interstitial fluid and may causean inflammatory reaction.

There is, therefore, a need within the transdermal sampling field for aminimally invasive sampling technique and apparatus suitable for rapid,inexpensive, unobtrusive, and pain-free monitoring of importantbiomedical markers and environmental toxin exposure. These propertiesand advantages of the present invention will become apparent to those ofskill in the art upon reading the following disclosure.

BRIEF SUMMARY OF THE INVENTION

The present invention pertains to a transdermal sampling system,comprising: at least one sampler for retrieving and transferring atleast one analyte obtained transdermally from the skin of a subject; atleast one detector system for identifying and quantifying said at leastone analyte; and at least one logic module for (i) receiving and storinginput data from said at least one detector, (ii) relating the input datato other data obtained from the subject, (iii) displaying outputinformation, (iv) transmitting the output information to another system,and (v) controlling the operation of said at least one sampler and atleast one detector.

The present invention also pertains to a microfabricated device forallowing remote monitoring of a subject, comprising: at least onesampler unit body for retrieving and transferring at least one analyteobtained transdermally from the skin of a subject; at least one detectorsystem connected to said at least one sampler unit body for identifyingand quantifying at least one analyte obtained from a subject; and atransmitter/receiver for transmitting data relating to at least oneanalyte detected by said detection system to a logic module forprocessing thereby, and for allowing control of the microfabricateddevice by a logic module

The present invention also pertains to a microfabricated device forsampling analytes from the skin of a subject, comprising: a detectionchamber for receiving analytes retrieved from the skin of a subject; aphotonic detection system, comprising a photonics source locatedattached to said microfabricated device in association with saiddetection chamber, and detectors associated with said detection chamberfor detecting analytes received in said detection chamber.

The present invention also pertains to a microfabricated device forsampling analytes from the skin of a subject, comprising: a detectionchamber for receiving analytes retrieved from the skin of a subject; apatch which changes color when contacted by predetermined analytes,located attached to said microfabricated device in association with saiddetection chamber; and detectors associated with said detection chamber,for detecting a change of color of the patch indicating the presence ofa predetermined analyte.

The present invention also pertains to a microfabricated device forsampling and detecting analytes retrieved from the skin of a subject,comprising: at least one conduit for retrieving and transmitting ananalyte from the skin of a subject to a detector; and means forenhancing permeability of the skin of a subject for retrieving said atleast one analyte therefrom.

It is an object of the present invention to provide a transdermalsampling system.

It is another object of the present invention to provide an integrateddetection system using patch type detector.

It is still another object of the present invention to provide anintegrated detection system using integrated photonics.

It is a further object of the present invention to provide amicrofluidic perfusion system for enhancing transdermal transfer ofbiological molecules.

It is yet an other object of the present invention to provide a thermalablation mechanism by resistive heating for removal of the stratumcorneum.

It is still another object of the present invention to provide a laserablation mechanism for removal of stratum corneum.

It is a further object of the present invention to provide amicrofluidic transfer of fluids utilizing capillary action.

It is another object of the present invention to provide an adhesive forholding transdermal sampling system on skin It is another object of thepresent invention to provide a chemical modification of channel surfaceswith antibodies containing fluorescently labeled antigens that areexpelled from the surface and detected down stream by competitivebinding.

A greater understanding of the present invention and its concomitantadvantages will be obtained by referring to the following figures anddetailed description provided below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of the overall architecture of themicrosystem of the present invention.

FIG. 2 illustrates in cross-section a single reservoir capillary pair.

FIG. 3 shows test results obtained for a back of the hand colorimetrictest for blood alcohol.

FIG. 4 shows the seal structure as viewed (a) from the bottom, and (b)in cross-section.

FIG. 5 is a cross-section of a device of the present inventionillustrating the non-invasive sampling sequence.

FIG. 6 is a cross-sectional view illustrating the sequence of operationof the Bio-Fluidic Integrable Transdermal (B-FIT) microsystem.

FIG. 7 schematically illustrates the basic fabrication steps for thethree main components of the system, shown in cross-section.

FIG. 8 is a schematic illustration of a detection scheme usingfluorescently labeled proteins or metabolites.

FIG. 9 illustrates in transverse section an alternative waveguide andsample chamber configuration.

FIG. 10 illustrates a B-FIT microsystem.

FIG. 11 illustrates a cross-sectional view of type C bed illustratingthe detection scheme.

FIG. 12 illustrates an overview of the ELISA microsystem informationalcomponent.

FIG. 13 illustrates a cross-sectional view of type CI showing themicrofluidic interconnect, coupling the external tubing with the siliconcapillary.

FIG. 14 illustrates a cross-sectional view of an alternative which usesa silicon sleeve around the DRIE capillary hole, showing the siliconsleeve microfluidic interconnect, coupling the external tubing with thesilicon capillary fabricated as wafer through-holes, and external tubingconnected to silicon capillary.

FIG. 15 illustrates a cross-section view of a third bed structureincorporating a collection chamber for the analyte, which has flowed upthrough DRIE capillary through-wafer hole by capillary action.

FIG. 16 illustrates a cross-sectional view of a fourth bed, designatedtype CIC, incorporating collection chamber and fluidic interconnect.

FIG. 17 illustrates the general fabrication process for the type CIarray, showing, (a) photoresist (PR) patterning for silicon sleeve, (b)oxide patterning of sleeve, (c) re-application of PR, (d) pattern forDRIE of bore hole, (e) remove PR and DRIE sleeve, and (f) remove oxide.

FIG. 18 illustrates a cross-section showing the double sided processingnecessary to fabricate the type CC (and type CIC) device.

FIG. 19 illustrates a magnified view of anchored spiropyrans in asilicon capillary.

FIG. 20 illustrates a single reservoir capillary pair.

FIG. 21 illustrates a single reservoir capillary pair.

FIG. 22 illustrates fabrication steps for wafer #2.

FIG. 23 illustrates retention volumes at varying concentrations of[3H]-EB.

FIG. 24 illustrates retention volumes at varying pH and ionic strengths.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides an enhanced system and method formonitoring the health of an individual and delivering drugs to anindividual transdermally. Specifically, the present invention providesan integrated, cost-effective, rapid and unobtrusive assessment of asubject medical condition. The invention further provides means fortransdermal delivery of drugs in response to the aforementionedassessment of a subject's medical condition. Embodiments include, forexample, monitoring a subject for pesticide exposure, monitoring thestress status of a war-fighter; phenotyping using the enzyme N-acetyltransferase to indicate an infected or diseased state; monitoringexternal exposure and internal contamination of a person with eitherorganophosphate nerve agents (tabun, sarin, soman) or organophosphateinsecticides (parathion and metabolites thereof); monitoringinflammatory sequeli in response to microbial infection (interleukin-1,interleukin-6, tumor necrosis factor); monitoring microbial toxins(anthrax, botulinum, endotoxin); monitoring spore metabolites arisingfrom human catabolism via lymphatic or hepatic pathways; monitoringstimulants such as caffeine, antihistamines (dexomethorphan, caffeine);monitoring stress through alterations in blood glucose concentration oraltered metabolism of insulin/glucose.

An overall architecture of a preferred embodiment of the presentinvention is shown in FIG. 1. The disposable B-FIT 100 is adapted todetect analytes of interest and is mounted in a receptacle 101 toprovide mechanical support and electrical connections, includingelectrical connections to the thermal heaters of the B-FIT. Theconnection receptacle 101 also accurately aligns the B-FIT with respectto a switchable photonic backplane. The connection receptacle alsopreferably contains a power source 102; logic control 103; andelectronic circuits for power management, electronic storage of results,electronic circuits for processing biochemical analysis data, electroniccircuits for timing events, and means for communicating results 104either directly or via telemetry. Optical components are provided,preferably located within B-FIT 100 or MEMS physiochip 106. In oneembodiment, fluorescence measurements are made sequentially upon each ofa plurality of analysis chambers contained in the B-FIT. Drug deliverychips 105 are also optionally provided by the present invention and areused to deliver potent drugs transdermally, for example, drugs used tocounteract nerve gas may be delivered. In addition, a physiochip 106 isoptionally provided that gathers continuous basic vital information,including blood pressure and pulse rate.

The transdermal subsystem, located within the B-FIT and drug deliverychip, functions to contact the skin with a physiologically compatiblesolution or a physiologically compatible solution containing a drug. TheB-FIT is organized into a dense array of somewhat independent singlereservoir capillary pairs. The capillary pairs each comprise a reservoircapillary 211 for retaining a physiologically compatible solution, thereservoir having a breakable seal 215 (illustrated in a ruptured state),and an adjacent transport capillary 212 for transporting physiologicallycompatible solution, which has contacted skin, to an analyte measuringsite. An adhesive layer 216 is provided upon the lower surface of theB-FIT. In use, the adhesive layer is interposed between the lowersurface of the B-FIT and the skin, and attaches the B-FIT to the skin.

In a preferred embodiment, a thermal perforation subsystem functions toablate a microscopic portion of the stratum corneum, the topmost layerof skin, so that the interstitium can be exposed. The thermalperforation subsystem is preferably comprised of a micro-heater in closeproximity to the skin surface, together with electrical components thatcontrol current to the micro-heaters.

A capillary array subsystem is preferably provided microfabricated intosilicon wafers that comprise the B-FIT. The invention preferablyprovides a plurality of capillary-array subsystems, each of whichcomprises a fluid delivery chamber or reservoir chamber 201 to deliver afluid to the skin surface, a capillary channel 202 to recover fluid fromthe skin surface, and at least one transverse capillary channel in whichthe analyte or analytes are detected. The B-FIT 200 is preferablycomprised of a multilayered assembly of micromachined silicon wafers: afirst wafer 204, a second wafer 206, and a detection layer 203. Thedetection layer preferably comprises a photonics system for visible orfluorescence measurements, or a layer that comprises colorimetricreagents that develop a color change in the presence of an analyte, orother means for detection of an analyte. The capillary subsystem thuspreferably comprises capillaries for storage, passage and analysis ofphysiological fluids. The diameter and surface coatings of thecapillaries are preferably optimized for controlling flow of the fluidand to prevent non-specific adsorbtion of fluid components onto thecapillary walls.

An optional integrated photonics system is provided by the presentinvention to determine, either qualitatively or quantitatively, thepresence of one or more analytes. The integrated photonics systemcomprises waveguides, lenses, mirrors, light sources, and lightdetectors. Preferably, the integrated photonics system is housed withinconnection receptacle 101, which is attached to a surface of B-FIT 100that faces away from the skin. In some embodiments, the integratedphotonics subsystem is replaced by a calorimetric analyte sensitiveregion, wherein a color change, perceived directly by an observer,indicates the presence of an analyte.

Each of these subsystems and the interactions between the subsystems isdescribed in greater detail below.

The B-FIT preferably contains an array of somewhat independent analytesensing devices, termed “single reservoir capillary pairs” 200. As usedherein, the term “physiological fluid” represents a fluid that isbiologically compatible with living tissue, and is, therefore,isotonically and otherwise physiologically (for example, pH) suitable asa medium for contacting, for example, viable epidermal cells or cells ofthe stratum corneum. An example of a physiological solution within thecurrent meaning is physiological saline solution. Each single reservoircapillary pair preferably contains a reservoir capillary 201 that storesand releases a physiological fluid to irrigate the skin surface or asmall region of the stratum corneum and recover analytes. A breakableseal 205 is preferably provided to control the timing of the release ofthe fluid to irrigate the skin. The fluid is preferably recovered into acapillary channel 202 that carries the fluid to an analysis location,for example a detection patch 203. The transdermal subsystem preferablyutilizes single reservoir capillary pairs to ensure that the analyte ofinterest, if present, is accessible to the fluid.

As the term is used in the present application, “transdermal dosimetry”refers to the collection and detection of trace quantities of analytesthat reach the surface of the skin by passive diffusion frominterstitial fluid underlying the outermost layer of skin, the stratumcorneum. It will be appreciated that, in one embodiment of the presentinvention, the interstitial fluid is sampled for the presence ofanalytes of interest. It will further be appreciated that, in anotherembodiment of the present invention, ablation of a microscopic portionof stratum corneum enables the physiologic solution from the reservoirto come into contact with the upper region of the underlying viableepidermis, enabling analytes in interstitial fluid to migrate into thephysiologic solution via passive diffusion for analysis.

“Non-invasive transdermal detection,” as the term is used in the presentapplication, means detection of substances below the skin that isachieved without physical or chemical modifications of the normal skinbarrier. Small molecular weight analytes that exhibit both water andlipid solubilities can be sampled by non-invasive techniques.

For example, sweat can be sampled from the surface of the skin andanalyzed for alcohol content by a colorimetric test indicative of bloodalcohol concentration, as illustrated in FIG. 3. In this example ofnon-invasive detection, alcohol is detected in sweat obtained from thebacks of the hands of seven male subjects who have ingested 0-4alcoholic drinks prior to the test. Alcohol contained in the sweatreacts with reagents contained within a reactive layer, resulting in aquantitative measure of alcohol content of the blood.

However, non-invasive techniques are not practical where the analyte hasa high molecular weight (for example, protein), is highly polar (forexample, glucose), or is poorly soluble. The outward flux of suchmolecules across the skin can be greatly enhanced by ablation of thestratum corneum. Ablation is performed to a typical depth of 30-60 μm,exposing the underlying viable epidermis, from which fluid can becollected and analyzed for analytes that only poorly penetrate unablatedstratum corneum. This technique is herein termed “minimally invasive”because only the stratum corneum is ablated while the underlying viableepidermis is not breached. In one preferred embodiment of the presentinvention, minimally invasive transdermal detection is achieved bymicroscopic heat ablation of the stratum corneum layer. In anotherpreferred embodiment of the present invention, minimally invasivetransdermal detection is achieved by laser ablation of the stratumcorneum layer.

An adhesive layer preferably provides an interface between the device ofthe present invention and the skin. The adhesive layer is affixed to thelower surface of the B-FIT assembly and functions to attach the B-FITassembly to a suitable portion of skin surface, thereby minimizingmotion of the B-FIT assembly relative to the skin for efficientsampling. Gaps in the adhesive layer are provided over each capillarypair to permit the physiological solution to contact the skin. Theadhesive layer prevents leakage of fluid laterally, and is preferablycomprised of a Band-Aid-type adhesive that is relatively waterimpermeable.

It will be appreciated that that portion of the B-FIT that interfaceswith the dermis preferably functions to firmly and occlusively place theB-FIT system in direct contact the external surface of skin (stratumcorneum) or uppermost region of the viable epidermis. Occlusive contactare preferably such that prevent lateral or vertical movement of theB-FIT from its initial position on the skin, that limit release of B-FITmaterials externally, and preclude entry of external materials. Movementpreventive properties include preferably an adhesive element locatedperipherally on the lowermost surface of the B-FIT and/or covering theentire B-FIT and adjacent skin surface. Additionally, the lowermostsurface of the B-FIT can be adhered to the dermis to prevent sheerforces that would displace the B-FIT from its initial position. Theocclusive nature of the attachment of the B-FIT to the skin serves toconfine all substances migrating from the body or skin within the B-FIT,including water vapor. This captured water vapor facilitates transdermalpermeation by hydrating the stratum corneum, rendering it more permeableto a wide variety of analytes or therapeutic drugs.

In one preferred embodiment of the present invention, minimally invasiveheat ablation of the stratum corneum is employed to achieve significantenhancement of the efflux of certain analytes. In preferred embodiments,thermal ablation is used to remove the stratum corneum over amicroscopic region of the skin through a mechanism of resistive heating.A micro-ablation unit containing a micro-heater is preferably fabricatedupon the surface of the B-FIT adjacent to each capillary pair, andprovides a conductive heat path to the stratum corneum. The micro-heaterpreferably comprises a pair of electrodes connected by a conductivepathway that is arranged, either by the use of a resistive material orby a serpentine conductive pathway, to provide sufficient resistance tothe flow of electricity such that an effective amount of heat isproduced so as to locally ablate an appropriate portion of the stratumcorneum. Electrical connections are also provided to each of the twoelectrodes to connect the micro-heating unit to a controller thatcontrols the application of an electrical current source to theelectrodes. In preferred embodiments, it is advantageous that themicro-heater protrude from the surface of the silicon substrate of theB-FIT to provide improved heat transfer to the stratum corneum andreduce the power consumption of the micro-heater. In one embodiment, aheat-sink material is incorporated on top of the micro-heater to directthe thermal flow towards the skin barrier rather than through the bulksilicon material. In another embodiment, the micro-heater is fabricatedonto a silicon mesa that protrudes from the main silicon substrate ofthe B-FIT. Such an embodiment may preferably require non-planarfabrication of electrical connections to provide conducting pathwaysfrom the silicon mesa to the contiguous bulk silicon substrate. Suchnon-planar fabrication techniques are known to those of skill in theart, as illustrated in Paranjape et al., Technical Digest, 1997International Conference on Solid-State Sensors and Actuators, Chicago,Ill., Vol. 1, pp. 397 (1997), herein incorporated in its entirety byreference.

The thermal ablation micro-heater is pulsed with a suitable alternatingor direct current to provide local ablation. Control of the duration andintensity of the heating pulse is preferably carried out to effectablation of the correct area and depth. The micro-ablation preferablyoccurs in a confined volume of the stratum corneum of approximately 50μm×50 μm×30 μm.

FIGS. 4(a) and 4(b) illustrate the seal structure as viewed (a) from thebottom and (b) in cross-section.

A physiological compatible solution that may or may not contain one ormore drugs is retained within the reservoir capillary 401 by a breakableseal 405 prior to use. The seal preferably provides an electronicallyaddressable means for opening the reservoir capillary and contacting theskin surface or exposed stratum corneum to the physiological solution.The seal comprises a closure at the bottom end of the reservoircapillary and a means for opening the reservoir capillary. In apreferred embodiment, the seal comprises a thin membrane 400 that ispreferably a dielectric bilayer that ruptures at elevated temperaturesand a metal conducting path. Preferably, any thin, non-toxic,membraneous material that is sufficiently tough not to tear prior tointended use, is not electrically conducting, and ruptures at elevatedtemperatures is a suitable material for use as the seal closure. Apreferred material is low-stress nitride. Control of the film stressesof the membrane is required during fabrication. Kinard et al:, IEEETrans. on Inst. Meas., 46(2), 347 (1998), which is herein incorporatedin its entirety by reference. To fabricate the seal, a metal conductingpath 402 is surface deposited upon a low-stress silicon dielectric 400.Preferred metals for microheating elements include evanohm. Since theheat used to rupture the seal is optionally also used to ablate the skinin certain embodiments, a careful balance of film stresses, thicknessand resistance is preferably achieved so as to provide both the desiredheating and rupture properties. Deposition of the metal upon the filmalso requires deposition of metal upon an irregular topography. Suchtechniques are known to those of skill in the art. Geist et al., NISTJournal of Research 95(6), 631 (1990), which is herein incorporated inits entirety by reference. The conductive path preferably terminates attwo electrical contact pads 403, 404, to facilitate passage ofelectricity through conductive pathway 402. In a preferred mode ofoperation, an electrical current passing through the thin conductivepathway heats the metal of conductive pathway 402 and causes therupturing of the underlying dielectric layer, thus, opening thereservoir capillary. It should be noted that an advantage of thispreferred embodiment of the present invention and this preferred seal,in particular, is that mechanical moving parts are absent, therebyenhancing reliability.

In certain preferred embodiments, the seal seals both the reservoircapillary and the capillary channel, and both are thereby openedsimultaneously.

FIG. 5 illustrates a B-FIT device cross-section, showing details on thenon-invasive/minimally invasive sampling sequence. An exemplary unusedcapillary pair 502 has an intact seal wherein the physiological solutionis retained within the reservoir capillary; upon application of asuitable electric current, the seal 501 is ruptured and thephysiological compatible solution first contacts the skin and is thenrecovered into the transport capillary; and finally a used capillarywith a ruptured seal 500 is illustrated. In this preferred embodiment,each capillary pair functions as a single-use unit so as to utilize theseal and physiological solution.

Similarly, FIG. 6 illustrates the sequence of operations of a minimallyinvasive embodiment of a B-FIT system to determine blood glucoseconcentration. A micro-heater 603 preferably operates to ablate aportion of the stratum corneum located below a gap in the adhesive layer604 at the same time, or immediately prior to, rupture of the seal 601.Such a device provides an “on-demand” analysis. A physiological solutionis preferably expelled onto the exposed viable epidermis and recoveredinto the transport capillary. The transport capillary preferablyconducts the solution to a detection patch where the glucose is detectedin a calorimetric reaction that produces a blue reaction. Note that inthis preferred embodiment, the current pulses delivered to the microheater and the seal may be the same or different; the heater and sealmay, therefore, be electrically connected either in series or parallel.

Capillaries within the silicon body of the B-FIT device can preferablybe fabricated by several techniques, for example by micro-machining, orby etching in place using deep resistive ion etching (DRIE) techniques.Referring now to FIG. 7, construction of a preferred embodiment of theB-FIT is illustrated. The device comprises three main parts: the mainbody 700 which is preferably made of silicon 702, and contains severalserpentine capillary channels 706, each with its own reservoir channel707; a bottom capping section 701 that forms the lower part of theserpentine structure and contains the micro-heating elements 703; and atop capping section 704, which forms the upper part of the serpentinechannel 706, and which optionally contains electrodes for assisting theflow of physiological fluids using electro-osmotic pumping through thehorizontal segments of the serpentine channel. The top-capping section704 is, in some embodiments, bonded to the main body: an advantage ofsuch an arrangement is good coupling of light into the capillary that isthereby achieved. The main body is preferably made of silicon. The mainbody 700 and the bottom capping section 701 are preferably permanentlyaffixed to each other to comprise a sensor 705, that can, in certainembodiments, be detached from the top capping section 704 after use andreplaced with a fresh array.

The reservoir and capillary channels are preferably fabricated within astandard silicon wafer. The dimensions of the capillaries are selectedto facilitate the transport of sweat, interstitial fluid, or otherphysiological fluid, out of the open end of the reservoir under theforce of gravity, and into a capillary channel through capillary action.In a preferred embodiment, the capillary channels are 25 μm in diameterand are approximately 500 μm in length, and the reservoir channels are50 μm in diameter but are etched slightly shorter than 500 μm in lengthto provide a back wall. A lateral portion of the serpentine capillarychannel 708 is provided, which provides for a region of fluid flow thatis parallel and adjacent to the upper surface of the main body of thedevice for optical detection of analyte. The lower inside surface of thelateral portion is optionally provided with a reflective surface, suchas a reflective metal coating, to facilitate optical detection. Thelateral portion of the serpentine capillary is, in a preferredembodiment, completed by a surface of the top capping section. In use,the transport of physiological fluids and the recovery of analyte isenhanced by rinsing the skin with fluid previously maintained within thereservoir channel and then recovering the same into the correspondingcapillary channel.

The surface of the capillary array system is preferably functionalizedto improve the properties of the surface, for example to preventadsorption of protein, and/or to attach biomolecules such as antibodiesto the surface. Molecules that bind specific analytes are used toimmobilize analytes for subsequent detection and quantitative analysis.Suitable biomolecules include, but are not limited to, antibodies,antibody fragments, artificial antibodies, lectins, hybridizable nucleicacids, nucleic acid binding proteins, proteins that bind nucleic acids,proteins that bind other proteins, proteins that bind cofactors,cofactors (for example, flavins, pterins, thiamine, pyridoxals,quinone), and other reagents that specifically bind biological analytes.

Capillary tubes are preferably modified by either chemical or plasmatreatment. This step aids surface cleaning of organic contaminants andintroduces surface hydroxyl groups on the capillary surface, which arepreferably reacted with a silane such as aminopropyl trimethoxysilane(APTS) to provide a free amine group as an anchor for coupling reagentssuch as antibodies. In a preferred embodiment, polyethylene glycol (PEG)silane derivatives are used to provide a surface coating that preventsadsorption of protein.

In one embodiment, a solution containing antibodies directed to ananalyte of interest is exposed to mildly oxidizing conditions known tothose of skill in the art, which provides aldehyde groups upon thesurface of the antibodies. The aldehyde functionality is then coupled toa free amine on the capillary tube surface via a Schiff base reaction,thus immobilizing the antibody to the capillary tube surface.

In a preferred embodiment, detection of the analyte of interest is bydone by means of fluorescence. A substance that is capable ofspecifically binding an analyte (for example, an antibody) 802 iscovalently attached to the surface of the capillary, as describedpreviously. The binding sites of the immobilized substance 802 arefilled with fluorescently labeled analyte 801, prior to use of theinvention. When analyte, 800, is present, it competes for the specificbinding sites, displacing a portion of the labeled analyte moleculesinto the solution. The degree of displacement of labeled analyte dependsupon the concentration of analyte in the solution. Therefore,measurement of the amount of fluorescence displaced into the solution,when suitably calibrated, provides a quantitative measure of theconcentration of analyte 800.

By preferably immobilizing a plurality of antibodies of differentbinding specificity, the binding sites of which are separately filledwith their respective analytes tagged with fluorophores with distinctemission and excitation spectra, multiple analyte determinations canpreferably be made within a single capillary pair. The use of spectralfilters and/or alternative light sources is used in a preferredembodiment to photoexcite and detect fluorescence from the differentfluorophores, and thereby, determine the contribution of eachfluorophore to the total fluorescent properties of the sample.

Preferred fluorophores for the present invention include rhodamines,fluoresceins, Texas red, Oregon green, Bidipy dyes, andaminonaphthalenes.

In one embodiment, N-acetyl transferase, isozyme 2, (NAT-2) activity ismeasured as a marker of adverse drug effects, toxicity andpredisposition to disease. The NAT-2 phenotype can detected, forexample, by detecting the ratio of two metabolites of caffeine producedby NAT-2,5-acetylamino-6-formylamino-3-methyl uracil (AFMU) and1-methylxanthine (1×). Utilizing the ratio of AFMU to 1×, the activityof NAT-2 can be determined. Polyclonal antibodies can be raised to thesetwo metabolites and then purified. These antibodies can also be used todetect AFMU and 1× in urine samples by ELISA.

In a preferred embodiment of the present invention, the reservoircapillary is provided with a micro-heating element located at theopposite end of the capillary to the seal. The micro-heater is activatedto provide local heating of the physiological fluid so as to produce abubble, thereby forcibly expelling the physiological solution from thecapillary once the seal is ruptured. Note that the micro-heaterfunctions as a pump means, but that the pumping is achieved withoutmechanically moving parts, thereby assuring increased reliability.

The micro-heating elements are preferably comprised of a resistiveconducting pathway deposited by conventional deposition methods upon thesurface of the silicon. Unlike the breakable seal, the heating elementsare designed to withstand elevated temperatures without destruction ofthe conductive pathway. The conductive pathway is, in one preferredembodiment, a serpentine pathway, in which a high-resistance pathway andlocalized heat generation are achieved through the use of a serpentinepathway comprised of thin conductive pathways densely arranged uponwithin a small surface area.

Another preferred aspect of the present invention is an integratedphotonics analysis subsystem. The integration of photonics componentsinto the B-FIT system permits increased density of assays, reduced size,lower power consumption, and decreased cost. In a preferred embodiment,the photonics components are housed within a plastic housing thatcomprises the top capping section of the device. Note that otherdetection methods are envisaged in the present invention and arediscussed below.

In such a an integrated photonics analysis subsystem, photonics sources,for example LED's or lasers, are combined with detectors, waveguides,couplers, and mirrors, to provide a fully-integrated optical detectionsystem for detecting analytes in the present invention. The photonicscomponents are preferably located upon, and attached to, the top surfaceof the main body of the B-FIT device in a top capping section.

Polymer waveguides with couplers for source and detector arrays arefabricated as integrated “flex circuits” for mounting. Fully integratedwaveguide structures are fabricated by means known to those of skill inthe art, such as monolithic fabrication of the waveguide by dry resistprocesses. −Low η waveguide material (η<η_(water)=1.33) is preferred.

FIG. 9 illustrates a preferred embodiment of a waveguide and samplechamber. In a preferred embodiment, capillary fluorescence is used todetect the analyte within the capillary. LED sources emitting green,blue, yellow, or red light, can be used to excite fluorophores. Thechoice of exciting wavelength is dictated primarily by the excitationspectrum of each fluorophore. In other embodiments, laser sources can beused to provide specific excitation wavelengths, although the cost,size, and power consumption of lasers is generally higher than forLED's.

In a preferred embodiment, the upper inner surface of the lateralportion of the serpentine capillary is completed by a surface of the topcapping section 900. Optical detection is preferably performed withinthe lateral portion. Light is conducted to and from the lateral portionby an integral waveguide fabricated within the, preferably plastic, topcapping section. The orientation of the waveguide runs parallel to thesilicon surface.

In another embodiment, the transverse capillary interrupts the path ofthe waveguide 903, so that the light conducted by the waveguide passesdirectly through a portion of the solution contained in transversecapillary 900. This embodiment has the advantage of simplicity: lensesand mirrors are not required to divert and collate the lightbeam.Fluorescence or absorbance measurements are preferably made within theportion of the transverse conduit that interrupts the waveguide. Apreceding conduit portion 902 preferably contains the binding reagentsthat give rise to the displacement into the solution of fluorophore whenanalyte is present. Subsequent conduit 901 preferably conducts thesolution out of the light path.

In an alternative embodiment of the present invention, lightmeasurements are made within a capillary that is constructed of amaterial having an index of refraction lower than that of water. Thisembodiment also eliminates the need for lenses and mirrors and offerssuperior signal to noise properties.

FIG. 10 illustrates a preferred embodiment of the B-Fit system platform.To facilitate the coupling of light from the waveguide 1008 into thelateral portion, and from the lateral portion into the waveguide, amicro-mirror 1005 is preferably provided. The mirror is integrated as apressed component in the top capping section, or is a separate componentplaced within the plastic housing by injection molding, or is fabricatedby any other appropriate means. Preferably the micro-mirror 1005 isoriented at approximately 45° relative to the silicon surface of thelateral portion of the capillary, and is positioned directly above thelateral section. A highly reflective surface coating, such as a metalcoating, is preferably deposited upon the surface of the mirror toreflect light from the horizontal waveguide downwards into the lateralportion of the capillary. A lens is preferably provided to collate thefluorescence excitation and emission light beams. Micro-lens 1012 is, inone embodiment, convex to provide divergence of the light beam enteringthe lateral section from the waveguide, and convergent with respect tolight leaving the lateral portion and entering the waveguide 1008. Inembodiments in which fluorescence detection is used, light from spectralregion capable of exciting the fluorophore is conducted along thewaveguide, strikes the divergent mirror and enters liquid containedwithin the lateral conduit. A fluorophore within the lateral conduit ispreferably excited and emits light of a longer wavelength. The emittedlight strikes the mirror, which converges the light, and re-enters thewaveguide.

Bandpass or notch filters may preferably be interposed in the light pathto optimize the signal-to-noise ratio of the detected fluorescence,depending on the bandwidth sensitivity of photodetector embodiment.

Light sources for the integrated photonics analysis subsystem includeLED's, which have recently become available in light-emission colorsfrom blue to green, thus essentially covering at least a portion of theexcitation spectra of most commonly used fluorescent probes. See, forexample, Fluorescent and Luminescent Probes for Biological Activity. APractical Guide to Technology for Quantitative Real-Time Analysis,Second Ed. W. T. Mason, ed. Academic Press (1999). Alternatively,microelectronic lasers can preferably be used where specific wavelengthsare required. Any light detection means can be used to detect theemitted fluorescent light. Photodiodes, phototransistors, Darlingtonpair phototransistors, or photoresistors can be fabricated onto thesilicon surface of the main body, or can be provided as separatecomponents.

Standard low power CMOS fabrication is preferably used to power themicrosystem, to provide sequential logic control, and to permit storageof data in memory and its manipulation.

It should be noted that, despite the foregoing disclosure offluorescence detection of analytes, the present invention is notrestricted to fluorescence measurements. Other detection methods thatare advantageously used in the present invention include, but are notlimited to, Raman, UV-VIS, and FTIR spectroscopy, includingtwo-dimensional techniques, and fluorescence correlation spectroscopy.Furthermore, radiation sensors and magnetic field sensors are alsouseful as the basis of detection in certain embodiments. For monitoringradiation workers and the like, a preferred sensor embodiment is anoptical random access memory (ORAM) material. These materials arecomposed of a photochromic molecule such as spirobenzopyram embedded ina poly(methyl methacrylate) matrix. The measurement approach is basedupon measurement of radiation-induced tracks in optical memory media.

An optical deflection magic field sensor is preferably utilized wheremagnetic field monitoring is desired. The microsensor comprises analuminum beam that is suspended above a micromachined silicon substrateusing four aluminum support arms. These arms hold the beam at its nodalpoints, which are points of zero displacement when the beam vibrates atthe fundamental resonant frequency. A sinusoidal current is forced toflow through one support arm, through the length of the beam, and outthrough the other support arm. The frequency of the sinusoidal currentis essentially identical to that of the mechanical resonant frequency ofthe beam. In the absence of a magnetic field, the beam is unaffected.However, in the presence of a magnetic field oriented perpendicular tothe beam, a magnetic force causes deflection of carriers, which in turncauses the beam to vibrate at its resonant frequency. The amplitude ofthe vibration is directly proportional to the magnetic field strength,which can be measured using a laser.

FIG. 10 illustrates the operation of an embodiment of the B-FIT systemwith respect to analyte detection. The physiological solution preferablycontacts the exposed viable epidermis following operation ofmicroheaters 1006 to ablate a portion of the stratum corneum, rupturethe seal, and expel the physiological solution from the reservoirchannel 1002. Solution containing analyte recovered from theinterstitial fluid bathing the viable epidermis preferably enters thecapillary channel 1004. Within the capillary channel, analyte displacesfluorescently labeled analyte from analyte binding molecules affixed tothe capillary walls. Displaced fluorescently labeled analyte ispreferably carried to the lateral portion where it is excited by lightconducted by the waveguide 1008, micro-mirror 1005, and micro-lens 1012.Light of a longer wavelength that is emitted by the fluorophore is, inone preferred embodiment, conducted back into the waveguide 1008 by thereversed optical pathway, and propagates to a detector.

The integration aspect of the present invention also preferably includesthe aspect that real-time monitoring of a subject permits the use ofadaptive control algorithms to optimize the conditions (for example,heating pulse characteristics, sampling rate, among others), and drugdelivery regimen, in response to data obtained. In this preferredembodiment of the invention, data machine-learning techniques arepreferably employed to derive or learn some function that relates onemeasure of the health of a subject to analyte measurements, therebypossibly acquiring the ability to predict the health measure fromsubsequent analyte measurements. Adaptive control algorithms utilized inthe present invention embody the steps of learning, adaptation,feedback, and decision-making. Since the body is a dynamic system, thesesteps occur simultaneously and continuously throughout the life of thedevice of the present invention.

FIG. 12 illustrates an overview of the ELISA microsystem informationalcomponent. A preferred aspect of the present invention is the largenumber of individual measurements that are possible over an extendedtime period. With extended periods of measurement, baseline drift mustbe accounted for so that significant deviations are accurately detected.The present invention preferably provides computational means foraccounting for baseline drift, and for thereby detecting deviations froma current baseline. This means is illustrated for an embodiment directedto monitoring health in a subject. With improved monitoring techniques,day-to-day variations in metabolism are preferably established in thehealthy individual, and limits set to detect early stages of infection,disease progression, and exposure to toxins.

The metabolism of exogenous compounds such as drugs is mediated by aseries of enzymes. The type and amount of these enzymes in eachindividual is reflected in the person's genotype and, based upon thegenetic information, individuals can be classified as more efficientmetabolizers (FAST) and others as less efficient metabolizers (SLOW). Inhealthy individuals, the relationship between genetic makeup (genotype)and its expression (phenotype) is conserved, i.e. FAST genotypes produceFAST phenotypes, while SLOW genotypes produce SLOW phenotypes. However,a disease state of the, individual can alter this relationship, as candiet, smoking, alcohol, environmental chemicals, and biological orchemical warfare agents, among other factors. The determination of aperson's NAT-2 genotype and the monitoring of that individual's NAT-2phenotype can be used as a direct and sensitive probe of heath andclinical status.

In this approach, polyclonal antibodies are preferably developed againstthe caffeine metabolites AFMU and 1×, and are used to determine NAT-2phenotypes in an embodiment of the present invention. Blood glucoselevels, cytokine levels, and dextromethorphan metabolite levels, canalso be monitored.

Machine-learning algorithms are preferably used to acquire a metabolicbaseline and to indicate when an individual's body begins to enter astate of distress or disease. The Winnow and Weighted-MajorityAlgorithms (Littlestone & Warmuth, Information and Computations 108,212, (1994) can preferably be used. These algorithms, withwell-understood formal properties, are capable of learning andperforming in non-stationary environments (i.e., in the presence ofbaseline drift).

The readings of the two caffeine metabolites, AFMU and 1×, arepreferably provided as inputs for computation, and the computationpreferably proceeds in two alternating and cooperative modes: a learningmode and a performance mode. In the learning mode, the device preferablycontinually calibrates itself to the wearer's body chemistry using anadaptive algorithm, which adjusts a set of weights, with the aid offeedback. User interaction is necessary only if the body is stimulatedin such a way that the levels of the metabolites are not indicative ofnormal body function (i.e., the user will provide feedback only forfalse-negatives). In the performance mode, the device preferably takesthe readings of the caffeine metabolites and, using the current conceptdescriptions (i.e., weights), makes a decision about the body's state ofhealth, which is then communicated to the user. Since the body is adynamic system, this process of learning, adaptation, feedback, anddecision-making preferably occurs continuously and throughout the lifeof the device.

The device also preferably acquires a model of the wearer's healthystate, and uses this model to predict states of health in the future.Formally, machine-learning methods derive or learn some function fn froma set of x, y pairs, such that y=f(x). Naturally, f( ) is anapproximation to the true function, which is unknown.

However, when levels of, say, troponin I begin to increase (suggestingan imminent heart attack), then the device will preferably need tosample more frequently, as the rate of change from one measurement tothe next will be increasing. In this situation, adaptive controlalgorithms are preferably employed, powerful enough to properly controlthe sampling rate, but simple enough to be realized in micro-hardware.

Thus, in a preferred embodiment, adaptive control algorithms can be usedto task the transdermal component to sample its wearer for the targetsubstances, and machine-learning algorithms can be used to acquire amodel, which may change, of the wearer's healthy state.

Referring back, FIG. 10 illustrates a preferred embodiment of a B-FITmicrosystem. This total modular system preferably includes: (1) thefluid transport system including reservoir channel 1002, and capillarychannel 1004; (2) micro-heater(s) 1006, (3) the photonics systemincluding waveguide 1008, micro-mirror 1005, and micro-lens 1012, and(4) the chemistry for analysis of selected analytes. The interstitialfluid containing molecules indicative of biomarkers are preferablyobtained using a minimally invasive technique employing controlledthermal micro-ablation of the stratum corneum. The micro-heater(s) 1006used for this are preferably incorporated directly into thesilicon-based subsystem that is part of the B-FIT microsystem. Foroptimal transport of interstitial fluids or analytes through theanalysis capillary, a second reservoir capillary, containing aphysiologically compatible fluid, is preferably used to drive all fluidstowards the upper surface of the module. The driving force is preferablyprovided by microheater(s) 1006 that produce bubbles to force the liquidto flow out of the reservoir capillary and over the thermally ablatedregion of the skin. Once the interstitial and physiological appropriateliquids containing tagged and untagged molecules reach the topholding-cavity, analysis can begin. The top of this total transdermaldetection platform can preferably be integrated with optical waveguides,comprising micro-mirror(s) 1005 and micro-lenses 1012 for properlydirecting the light within the holding-chamber. The light that strikesthe analysis region of the holding-cavity is used to excite thefluorescently tagged molecules. The intensity of this fluorescence ispreferably picked up through the return path by the same opticalwaveguide.

The modular nature of the microsystem provides an excellent platformthat can be easily adapted for many innovative applications by applyingnew chemistries for the detection of selected analytes. For example, oneanalyte or biomarker that is especially important to children exposed topesticides is acetylcholine. Acetylcholine is located throughout thebody and when it is released, it acts as an excitatory neurotransmitterto propagate nerve conduction in the peripheral and central nervoussystems, or to initiate muscle contraction. Exposure to organophosphoruspesticides causes inhibition of acetylcholinesterase activity resultingin an accumulation of acetylcholine. This increase in acetylcholineconcentration will act as a biomarker, measured using the device byfirst establishing a baseline in an unexposed child. The MEMS-basedpatch is small and unobtrusive, permitting a child to live his/her dailylife while being continuously monitored for exposure to pesticidecontamination and providing early warming diagnostics.

Thus, one embodiment of the portable biomedical monitoring device of thepresent invention is as a pediatric micro patch system (PEP). Inproviding such a PμP device there are three tasks. Task 1 is thefabrication of silicon bed structures that function analogous to theB-FIT Microsystem. As described above, the bed functions to deliverfluids to the interior of the capillaries and to the collection chamber.In addition, the bed mirrors the B-FIT microsystem with regard to theintegration of the chemistry. Task 2 is the chemistry to detectacetylcholine. This task includes chemically modifying a flat sample ofsilicon, which enables a functioning method for integrating chemistry tothe bed. Task 3 involves the testing and validation phase, where thechemistry protocol is adopted for the capillary bed. Detection limits ofacetylcholine are established and sample bodily interstitial fluid istested.

With regard to the B-FIT system, the fabrication of the capillary bedstructures relies, in one embodiment, on bulk micromachining of silicon,accomplished through either deep reactive ion etching (DRIE) or wetchemical etching. The DRIE process preferably enables the fabrication ofhigh aspect ratio through-wafer holes that form narrow micro-capillariesof varying diameters. Wafers with a nominal thickness of 500 μm areused; however, a preferred thickness can be established through surfacemodification testing.

For the exemplary bed structure shown in FIG. 10, (type C), an array ofcapillaries with varying diameters are preferably formed usinglithographic patterning and DRIE. This type of structure enablesselection for the optimal dimensions required for capillary action toallow liquids to be drawn up and inside the channel, because wetchemistries are involved in both capillary-wall surface modificationsand during fluorescence validation using a test solution containingacetylcholine. Once the capillaries have been chemically modified,testing for fluorescence is preferably accomplished using a laser sourceat the top-side entrance port of the silicon capillary, and a detectorlocated at the exit port on the bottom-side.

Returning to FIG. 11 illustrating this preferred detection scheme, thespot size of the laser light path 1102 can preferably be adjusted tomatch the diameter of silicon capillary hole 1104 etched in siliconsubstrate 1106, while its excitation wavelength is preferably held at430 run, to match the frequency required to excite the fluorophorecausing it to emit fluorescent light 1108. The detector preferablyincludes photomultiplier 1110, and monochromator 1112 is preferably usedto tune the detector to the fluorescence wavelength of 567 nm. Inaddition, notch filter 1114 is preferably used to greatly attenuate theunwanted laser light frequency from reaching the photomultiplier.

A second exemplary bed structure, termed type CI, is similar to thebasic capillary array with a modification to the entrance port that isfitted with a microfluidic interconnect. This design is preferably usedas an alternative to type C, in a situation where capillary actionperhaps does not function appropriately. In such circumstances, type CIprovides an interconnect mechanism allowing for external tubing orsyringe ports to be directly coupled to the silicon capillary forsurface modification and testing purposes.

FIG. 13 illustrates a cross-sectional view of a type CI bed structureshowing the microfluidic interconnect, coupling the external tubing withthe silicon capillary. Typically, a hole produced by DRIE can preferablybe made so that its inner and outer diameters match that of theinterconnect tubing, which is inserted into the opening and held inplace with adhesive 1302. Thus, DRIE microcapillaries fabricated aswafer through holes 1304 are in silicon substrate 1306. The holes arepreferably produced such that the inner and outer diameters match thatof external tubing connected to silicon capillary 1308, wherein thetubing is held in place with adhesive 1310. However, care must be takensuch that the adhesive used to hold the tubing does not seep into thecapillaries blocking the flow.

FIG. 14 illustrates a cross-sectional view of an alternative embodimentwhich uses a silicon sleeve around the DRIE capillary hole, showing thesilicon sleeve microfluidic interconnect, coupling the external tubingwith the silicon capillary. The sleeve preferably provides enhancedmechanical integrity for the external fluidic component, but alsoprevents adhesive 1402 from seeping and plugging the capillary hole.Once the external tubing is attached to the silicon substrate, chemicalsand analytes can preferably be injected using either pressure gradientsor syringes. The external tubing is then removed, having fulfilled itspurpose of introducing fluids into the narrow capillary channel, and theverification procedure to detect fluorescence can start, as for the typeC device. FIG. 14 thus shows adhesive 1402, silicon substrate 1404, DRIEmicrocapillaries fabricated as wafer through-holes 1406, and externaltubing connected to silicon capillary 1408.

FIG. 15 illustrates a cross-sectional view of a third bed structureincorporating a collection chamber for the analyte, which has flowed upthrough DRIE capillary through-wafer hole 1502 by capillary action. Forthis microstructure, called type CC, the silicon capillary is preferablyfabricated using DRIE followed by an anisotropic wet silicon etch tocreate the collection chamber on the front-side of silicon substrate1504. With this bed, it is sufficient to chemically modify only thesurface of this chamber. The analyte preferably flows up the capillarychannel and reacts with the immobilized chemistry in the chamber toproduce fluorescence light path 1506.

FIG. 15 also illustrates preferred excitation laser 1508 andfluorescence light path 1510 detection set-up. The excitation anddetection method preferably makes use of excitation laser 1508,photomultiplier 1512, and monochrometer 1514, respectively, as before,however, now the setup is preferably only on the front-side of the bedstructure. The photomultiplier 1512 and monochrometer 1514 arepreferably set directly above the collection reservoir to act as thefluorescence detector. In this situation, the impinging laser light canpreferably be directed towards the collection chamber at an angle suchthat its reflection does not contribute to photomultiplier 1512detection. Nevertheless, notch-filter 1516 can preferably be usedbetween the detection unit and the chamber to eliminate any stray lightfrom excitation laser 1508.

FIG. 16 illustrates a cross-sectional view of a fourth preferred bed,designated type CIC, incorporating collection chamber 1602 and fluidicinterconnect. In addition to the silicon capillary channel andcollection chamber of type CC, this bed preferably also includes afluidic interconnect mechanism on the back-side of the wafer. As before,this design can preferably serve as a fallback mechanism if thecapillary action does not provide enough capillary force to draw thefluid up to the collection chamber. The fluidic interconnect preferablymakes use of the silicon sleeve 1604, as described in the type CItest-bed structure. DRIE capillary through-wafer hole 1606 and siliconsubstrate 1606 are also shown.

For each variation in bed structure, fabrication of the through-wafercapillary arrays is made on single-sided polished, <100>-type 4 inchsilicon wafers. The array of capillary holes preferably consists of fourdiameter values (25 μm, 50 μm, 75 μm, and 100 μm) with a nominal lengthof 500 μm, which corresponds to the wafer thickness. For the type Cdesign, the patterns for the holes are preferably formed in aphotoresist layer, which acts as an ideal masking layer to the DRIEprocess. A single (standard) photolithographic step preferably producespatterns on the front-side of the polished silicon surface. Although theDRIE process renders an anisotropically etched cavity, some undercuttingof the mask takes place. Thus, the pattern of the mask takes intoaccount this unavoidable lateral etch to achieve the desired diametersfor the capillaries. Depending on the type of DRIE system used, theratio of vertical-to-lateral etch is better than 50-to-1. That is, forevery 50 μm of etch depth, there is approximately 1 μm of under-etchbeneath the masking layer. The masking dimensions are thereforedependent on this etch parameter, which can be determined through priortesting. DRIE services can be obtained, for example, through one of theNational Nanofabrication Facilities, or through the MEMS Exchangeprogram.

FIGS. 17(a) to (f) illustrate a preferred general fabrication processfor the type CI array, showing, (a) photoresist (PR) patterning forsilicon sleeve, (b) oxide patterning of sleeve, (c) re-application ofPR, (d) pattern for DRIE of bore hole, (e) remove PR and DRIE sleeve,and (f) remove oxide. The initial step, prior to lithography, is to growa thin layer of thermal silicon dioxide over the entire silicon surface.Photoresist is applied, and the oxide is patterned and etched todelineate the locations of the silicon sleeves that are located aroundeach capillary hole providing for microfluidic interconnections. Thisstep is followed by another application of photoresist, and thecapillary locations are patterned into both photoresist and oxide.Through-wafer holes are again formed using DRIE, as with the type Cdevice. The photoresist is subsequently removed, leaving thepre-patterned layer of thermal oxide on the front-side of the siliconsurface. A much shorter DRIE step is performed, with the oxide layeracting as the masking layer, to create the silicon sleeve.

For both type CC and CIC the fabrication process preferably requires thewafers to be double-sided polished since back to front alignment isrequired. For the type CC device, the collection chamber is preferablybulk micromachined into the front-side of the wafer using an anisotropicwet chemical etchant. Subsequently, a thin thermal oxide is grown onlyon the front-side to act as a passivation layer, while the back-side iscoated with photoresist. The DRIE procedure for the capillaries isperformed from the back-side so the capillary hole aligns with one sideof the collection chamber.

FIG. 18 illustrates a cross-section showing the double sided processingnecessary to fabricate the type CC (and type CIC) device. The wafer isthen inverted so that subsequent processing of the capillaries andsilicon sleeve interconnects on the back-side follows the same procedureas that for the type CI device.

With regard to a preferred surface modification aspect of the presentinvention, the technical approach to providing a surface boundfluorescent probe specific for the biomarker acetylcholine is preferablyaccomplished by modifying the method developed for liquid phasedetection set forth in Inouye, M. et al., Nondestructive Detection ofAcetyl Choline in Protic Media: Artificial Signaling AcetylcholineReceptors, J. Am. Chem. Soc., 116, 5517 (1994). In a preferredembodiment, the method utilizes spiropyrans, which are inexpensive andreadily available from commercial sources. They are known for theirspectral properties and are very robust, especially compared withmolecules used for standard ELISA detection methods. The spiropyrans aresynthetically surface immobilized on the silicon bed, in either acollection chamber or in a capillary, using silane chemistry andstandard coupling chemistry.

FIG. 19 illustrates a magnified view of anchored spiropyrans in asilicon capillary. A spiropyran (for example,C-methylcalix[4]resorcinarene) is preferably modified to incorporate acarboxylic acid cross-linking group that can be coupled to a freeamino-silane modified silicon surface. Stoichiometric addition of baseto the spiropyran allows for the reaction of an ω-bromocarboxylic acid(for example, 5-bromopentanoic acid). The length of this molecule isrelated to its solubility and reaction efficiency. Longer carbonchain-lengths are more soluble, but harder to couple to the surface,while longer carbon chain-lengths are less soluble and more likely tocouple to the surface. The synthesis can be followed by NMR spectroscopyas needed to examine and characterize reaction products.

The spiropyran or resorcinol/acetaldehyde tetramer preferably forms atetraphenolate in akaline media that arranges in a bowl shaped cavityand can complex alkylammonium cations. When complexed with a pyrenemodified N-alkyl pyridinium cation (PPC), no fluorescence is observed.The PPC may be purchased or synthesized depending on the selectedmethod. One preferred method of incorporation of PPC is by solutioncomplexation with the spiropyran. After anchoring the spiropyran, PPC isintroduced and the complex is formed. The competitive binding ofacetylcholine kicks off PPC and produces a fluorescent complex. Thiscomplex is detected using the laser/detector scheme described above inthe microfabrication approach. PPC can also be incorporated by formingmixed monolayers of the spiropyran and the PPC. In this way, complexesare formed at the solution surface interface. Another preferred way ofmaking PPC complex with the spiropyran is by synthetically attaching PPCto the spiropyran as describe by Inouye et al., supra. This methodallows for intramolecular quenching of the fluorescence as opposed tointermolecular quenching as described in the first two methods.

Upon completion of synthesis, silicon substrates are preferablyderivatized with silanes such as 3-aminopropyltrimethoxysilane. Thereaction provides a free amino group on the silicon surface that can becoupled using a water-soluble carbodimide, such as EDC, to thecarboxylic acid of the modified spiropyran. X-ray photoelectronspectroscopy (XPS) and contact angle measurements can be employed toanalyze the progress of varying surface attachment reactions. In apreferred embodiment, the highest surface coverage is achieved. Inaddition to surface coverage, the fluorescence efficiencies is examinedusing a fluorescence microscope. This aids in qualifying the activity ofthe attached spiropyrans. A simple experiment monitoring the qualitativefluorescence intensity before addition of acetylcholine and after theaddition of acetylcholine provides a baseline. At this point, the methodis transitioned into bed devices for testing.

During the microfabrication task, DRIE and wet chemical etch rates arepreferably determined using test samples in order to produce the properbed structures. In addition, preferably samples are cleaved and viewedthrough a scanning electron microscope to determine whether the propercross-sectional geometry of the through-wafer capillaries has beenachieved. For the chemistry task, surface modification and chemicalsynthesis is preferably used to validate the immobilization protocol ona flat sample of silicon. This determination requires the detection offluorescence after excitation on a relatively large sample; thus, afluorescence microscope is used during this testing procedure.

Once the chemical synthesis and surface modification tasks are completedthrough rigorous large sample testing, the chemistry is then tested onthe small-scale capillary bed structures. For this phase, aphotonics-based test set-up is preferably employed based on theexcitation and emission of fluorophores. Excitation is through directabsorption from an external laser source. Several preferred sources areavailable for various testing strategies, including tunable continuouswave (CW) argon ion pumped dye laser, an air-cooled argon ion laser, aNd:YAG nanosecond pulsed laser which pumps an optical parametricoscillator, and several smaller HeNe lasers with both red (632.8 nm) andgreen (543 nm) wavelength outputs. The Ar ion pumped dye laser hasoutputs from the pump laser at wavelengths of 488 nm and 514 nm, with amaximum power of 9W. The maximum power from the CW dye laser is 3W, andis tunable in the ranges of 590 nm. to 600 nm and 610 nm to 630 nm, withan additional output at 577 nm. The air-cooled Ar ion laser has a singleoutput at 514 nm and provides approximately 70 mW of power. The Nd:YAGlaser has a fundamental wavelength of 1064 nm, along with the doubled(532 nm) and tripled (3.55 nm) outputs achieved with internal harmonicgenerators (KDP crystals). The pulse width is 5 to 7 nanoseconds, withpeak pulse powers of over 200 mJ. However, since the fluorophore beingimmobilized on the silicon surface needs to be excited at 430 nm, thepower from the tripled output Nd:YAG laser can be used to pump anoptical parametric oscillator (OPO), based on a beta Barium boratecrystal (BBO). This is essentially a resonant optical cavity containingthe nonlinear BBO crystal. The pump beam is converted to the so-calledsignal and idler beams, where the wavelengths following the relation:${\frac{1}{\lambda}\quad{pump}} = {{\frac{1}{\lambda}\quad{idler}} + {\frac{1}{\lambda}\quad{signal}}}$which arises from the photon energy conservation requirement. The ratiobetween the two output wavelengths is governed by the angle of the BBOcrystal with respect to the incident beam. Using this, the outputwavelength can be tuned by changing the crystal angle. Either the signalor idler output can be eliminated using a high- or low-pass opticalfilter at the output port of the OPO. This output is tunable indifferent ranges from about 400 nm. to 2200 nm. The ranges are set bythe resonant cavity mirror properties and the output filters. Peak pulseenergies in these ranges are on the order of 10 mJ.

The output fluorescence is preferably detected either in the forwarddirection or at some angle depending on the microstructure geometry. Ineither case, the incident laser light is preferably blocked using both aholographic notch filter and a monochromator. The notch filter, commonlyused in Raman spectrometers, cuts all light at the wavelength of theexcitation light, whether coming directly from the laser or fromRayleigh scattering. The monochromator provides further rejection ofunwanted light, both ambient and from the laser. The monochromator alsoallows tuning to the maximum of the emission spectrum, for optimizationof the signal-to-noise ratio. Finally, detection is preferablyaccomplished using a photomultiplier and a boxcar-integrator detectionscheme, with gating from the laser electronics. Alternately, the signalis detected with a silicon avalanche photodiode, providing higherdetection efficiency.

With the type C bed, capillary action is preferably tested in order to,first, modify the internal silicon surface wall of the capillary hole,and then, second, to draw up analyte, which reacts with the immobilizedsidewall chemistry. If the capillary forces are not sufficient to drawup significant amounts of fluid for the capillary dimensions beingtested, then the type CI test-bed is preferably used. This allows directphysical insertion of fluids within the capillary using a pressuregradient or a syringe pump connected to the microfluidic interconnects.In either case, the photonic detection system is preferably used to testthe feasibility of performing in-capillary fluorescence measurements. Incontrast, the type CC test-bed requires chemical modification onlywithin the silicon surface of the collection chamber. Therefore, thisdevice can also be used to test capillary action as well as the photonicdetection system situated on the front-side of the wafer. Again, thedevice can preferably be tested physically by inserting the analyte intothe capillary array, using bed type CIC.

Once a particular bed is selected, further testing preferably relates todetermining the selectivity and sensitivity of the biomarker to theimmobilized chemistry within the capillary array. This type of testingis preferably conducted, for example, by introducing a solutioncontaining acetylcholine at varying concentration levels. By determiningthe amount of fluorescence variation, using the output from thephotomultiplier, with corresponding changes in acetylcholineconcentration levels, a quantitative indication of the lower limit ofdetection of acetylcholine, and therefore device sensitivity, isobtained. To determine selectivity, another series of tests arepreferably performed to introduce other neurotransmitters in variousconcentrations within the analyte solution and to determine theirrelative fluorescence with respect to that obtained for acetylcholine.The more common neurotransmitters that can be used in this testingphase, other than acetylcholine, include adrenaline, dopamine,serotonin, tryptamine, histamine, and glycine. For comprehensive testingof selectivity, the device is preferably tested for otherneurotransmitters such as noradrenaline, tyramine, glutamic acid,aspartic acid, taurine, and proline that could introduce an unwantedcross-sensitivity fluorescence response. Finally, in vitro testing ofthe device preferably using traditionally extracted interstitial fluidfrom human donors is conducted. Samples are taken from individualsexposed to high levels of pesticides, and a control set is taken fromthose who were not exposed to toxic environments.

A sample result of this fabrication and testing process is preferably asilicon-based capillary array bed (PμP) with a chemical immobilizationprotocol for the detection of acetylcholine, which is a biomarker foroganophosphate type pesticide exposure. In addition, a sensitivityprofile is established. The PμP microstructure design allows it to bereadily interfaced and integrated as a module to the B-FIT transdermalsampling platform. The transdermal sampling process is preferablyinitiated using a minimally invasive micro-thermal ablation heater toreach the stratum corneum/viable epidermis interface, allowing forextraction of interstitial fluid. The B-FIT microsystem makes use ofsilicon fabricated capillary arrays to allow for interstitial fluidtransport to a glucose-sensing patch situated on top of the array. Thebasic capillary array structure of the PμP microdevice can beincorporated into the B-FIT. The detection mechanism for the biomarkeracetylcholine in PμP preferably consists of the synthesis of afluorescent spyropyran that is surface immobilized. With the chemistryidentified, surface modification of the bed is carried out, allowing forease of manufacture of low-cost, minimally-intrusive chip scaledetection.

The chemistry developed relies upon an alternate preferred embodiment ofthe B-FIT microsystem device, incorporating a photonics componentinstead of a glucose patch. This system is preferably fitted withwaveguide technology on the top of the array, which is used to transmitand detect excitation and fluorescence light, respectively. Again, thePμP microdevice is ideally suited as a module to the B-FIT, in view ofthe fluorescence detection of acelylcholine. Testing of simulated bodyfluid and human serum can preferably be done on the test bed todetermine the sensitivity and the specificity of the chemistry.

The present invention allows for the fabrication and chemicalimmobilization of any number of biomarkers, thereby creating a set ofmodules to be “plugged into” the B-FIT platform. Numerous examples,possibilities, and applications exist, ranging from a vast number ofmolecular biomarkers for health monitoring through enzyme and metabolitedetection, to hormones. For pesticide detection some of the other keybiomarkers would be acetylcholinesterase, acetic acid, and choline. Itis also important to detect other analytes, aside from organophosphates,made possible through the use of the PμP microdevice. These includeanticholinesterase insecticides (phosphorothionates), organochlorineinsecticides (DDT, Dieldrin, Lindane), pyrethroid insecticides(Permethrin, Fenvalerate), herbicides (TCDD, Paraquat), and rodenticides(Warfarin, Diphacinone, sodium fluoroacetate, strychnine). Other keybiomarkers to trace would be the antidotes such as atropine andpralidoxime.

Processing steps and the respective equipment in the fabrication of thePμP preferably include the following: (1) lithography: a front-side maskaligner capable of 1 μm line resolution with UV and deep-UVphotolithography; a fixture capable of two sided alignment; aphoto-resist spinner, pre- and post-bake ovens and associated processingchemicals; (2) deposition: a magnetron sputtering system capable ofdepositing metals (Al, W, Ni, Ti, Pt, etc.), and magnetron reactivesputtering of oxides which can be provided in the fabrication; an e-beamevaporator with three hearths for low energy deposition of metals; anddeposition apparatus for PECVD oxides and nitrides for coating surfacesto adjust for stresses and adhesion; (3) film treatment, to adjust thestresses and strengths of films and membranes using rapid thermalannealing capability; (4) photo-mask design and fabrication; (5)etching: deep reactive ion etcher (DRIE), RIE equipment and wet TMAHetching; (6) diffusion and heat treatment: high temperature furnacescapable of wet and dry oxide growth and furnace soak annealing which canbe required for heaters comprised of dolled silicon; and (7)measurement: a thin film stress tester and a Leitz thin film analyzer ora Nanometrics Automatic Film Thickness measuring apparatus for measuringfilm thickness. Microscopic examination is available with a high qualityLeitz microscope and a Zeiss SEM with EDS.

The transdermal transfer system (TTS) is preferably manufactured usingvarious standard processing and fabrication technologies. The TTSmicrodevice fabrication also relies on several micromachining steps,from simple bulk micromachining to deep reactive ion etching (DRIE)procedures.

The fabrication process steps of the TTS microdevice preferably involvesilicon processing of two wafers, as indicated in FIG. 20. Wafer #1preferably comprises the reservoir channel, capillary channel,micro-ablation unit, and breakable seal. The micro-ablation unitcontains the micro-heater along with a heat-sink to provide a highlyconductive thermal path towards the stratum corneum. By incorporating aheat-sink on the micro-heater, the heat transfer is more favorablydirected towards the stratum corneum. Wafer #2 preferably contains thereservoir micro-heater, which is preferably aligned to mate with the topof the reservoir channel.

FIG. 21(a-e) provides cross-sectional fabrication diagrams of the waferprocessing steps for wafer #1. Double-sided polished, 300 μm thicksilicon wafers are preferably used in the processing steps becauseworking will be done on both the front and back sides. Initially, in oneembodiment, the microablation heater is formed by depositing andpatterning a metal layer onto a patterned silicon dielectric layer toform a serpentine heating element through which current is passed. Thedielectric is preferably patterned as square region where the heatingelement resides. A preferred heater material can be selected throughthermal simulation. In addition to the heating element, a temperaturesensor can preferably be integrated alongside to monitor the localtemperatures generated by the current through the heating coil. This isindicated in FIG. 21 a, where processing occurs on the top-side of thewafer, but will eventually be inverted to become the bottom. In thisversion of the design, all bonding pads and traces are located in theplane of the heating element. The metallic traces and heating elementsare preferably insulated and protected by depositing a layer oflow-stress silicon nitride across the wafer. Although stress-freenitride is not required for this passivation purpose, it can find anapplication in the subsequent step.

Referring back to FIG. 4(a-b), the second step is preferably tofabricate the breakable seal. The seal is preferably composed of abilayer formed by the low-stress silicon nitride layer, deposited in theearlier step, and a metal that can weaken at elevated temperatures. Thearea where the metal is deposited determines the location of thereservoir capillary. Since the capillary dimension is preferably on theorder of 75 μm, the breakable seal must be situated in this 75 μm regionin order to open the reservoir capillary. FIG. 4 shows the bottom viewdesign of the breakable seal. It consists of two low-stress siliconnitride flaps bridged by the seal metal. By passing large enoughcurrents through this metal strip, the heat is sufficient to weaken themetal seal thereby releasing the nitride flaps. Although the nitridelayer is low in stress, preferably there is some controllable tensile orcompressive strain. By adjusting the deposition conditions of thesilicon nitride, it can be made in slight compression so that when theseal ruptures, the nitride flaps crudely act as unidirectional valves.

The preferred third major processing step is to form the heat-sink ontop of the micro-ablation heating element. As discussed above, aheat-sink preferably directs the heat towards the stratum corneuminstead of within the bulk silicon material. Without the heat-sink, themajority of the heat travels through the silicon, because its thermalconductivity is higher than air. By depositing an aluminum heat-sink onthe heater, the resulting heat flow is approximately divided evenlybetween silicon and aluminum. This is because the thermal conductivitiesof both silicon and aluminum are comparable, but by selecting a metalwith a higher thermal conductivity than that of silicon and aluminum, amore efficient heat transfer can be achieved. In a preferred embodiment,aluminum is used as the heat-sink material, however additional materialscan be applicable. In addition to increasing thermal flow towards thestratum corneum, the placement of a heat-sink preferably reduces theoverall distance between the source of heat and the skin barrier,thereby reducing power consumption. The aluminum is preferably patternedusing a lift-off procedure. However, since a thick metal layer may beneeded, preferably either a thick photoresist is used or the aluminum isdeposited over the entire wafer. This may cause problems with the metalseal, therefore a thin protective isolating layer is preferablydeposited prior to the aluminum. The aluminum is then lithographicallypatterned after the fourth step, to remain only on the micro-ablationheater. The patterning is done at a later stage in order to keep thesurface planar for subsequent processing steps.

The fourth preferred processing step involves inverting the double-sidedpolished wafer to reveal the as yet unprocessed side. A photoresistmasking layer is preferably deposited in order to pattern the openingswhere both reservoir and thin capillaries are formed simultaneously.These capillaries are both fabricated using deep reactive ion etching inorder to obtain narrow, high aspect ratio through-wafer holes. The thincapillary is preferably designed to be 25 μm in diameter while thereservoir capillary is about 75 μm in diameter, both with nominallengths of 300 μm. During the DRIE process, the silicon isanisotropically etched until holes are made through the wafer. However,for the reservoir capillary, the etching process terminates on thesilicon-nitride that is already present on the wafer backside. This isbecause the nitride acts as an etch-stop for the DRIE etch process. Asall processing of wafer #1 is now complete, the aluminum heat-sink canbe defined and the isolating layer can be removed.

The preferred processing steps for wafer #2 can also be outlined by across-sectional fabrication diagram, as shown in FIG. 22. The sequenceof steps is far less laborious, however some alignment issues stillexist. The first step in this preferred process is to fabricate thereservoir heating element on the front side of the silicon wafer. Thisis preferably done, as before, by depositing a heater material onto asilicon dielectric surface. The material is preferably patterned in theform of a heating coil, and is subsequently covered by a protectivesilicon nitride passivation layer. The preferred next step is to depositon the surface an etch-stop dielectric layer. Next, the wafer isinverted and patterned using DRIE to form the connecting capillaryopening. Once complete, the third step preferably involves depositing alayer of silicon dioxide onto the side containing the reservoir heater.This allows for the final step, anodically bonding wafer #1 with wafer#2. Care and attention preferably is taken to ensure that the reservoirheater mates with the reservoir capillary opening, and also to ensurethat the capillary from wafer #1 connects properly with the capillaryformed in wafer #2.

Each capillary and reservoir pair is preferably addressed individuallyso as to expose only one such pair to the skin surface in order toperform a single fluid analysis. Once employed, the open end of thecapillary continues to remain exposed to the skin, but is not addressedfor any further use.

A preferred embodiment includes additional considerations regarding thetiming for signals to open seals and control heaters. The total amountof energy imparted to the heaters that affect the ablation of thestratum corneum and the time over which that energy is imparted are alsoconsiderations. The system is designed and tested for minimal ablationenergy. That is, the minimum energy for the minimum duration is asignificant parameter for operation, resulting in minimal damage of theunderlying viable epidermis, and therefore minimizing the invasivenature of the process.

Other timing issues are also considerations, including the timing of theablation process (heaters) with relation to the opening of a capillaryseal. During the time the seal for a given capillary is being ruptured,the micro-ablation heater is preferably pulsed with an appropriatealternating current to thermally remove successive layers of the stratumcorneum.

Another timing consideration is the heater pulses associated with thereservoir erriptying process. The timing of heater pulsing is aconsideration to keep the reservoir flowing, and not taking up fluidfrom the stratum corneum. The heater at the top of the fluid reservoirpreferably forces out the liquid contents. Control of this heaterpermits control of the flow of liquid during the reservoir/capillaryanalysis lifetime.

Preferably tests of the various subsystems are done to establish thedermatoxicological and clinical pharmacological advantages. The testsequence is preferably sequential, starting with simple tests on variousmaterials, progressing to in vitro tests on human cadaver skin or animalskin, then to complete animal testing and finally clinicalpharmacological testing with human subjects. One shot valves coveringthe capillary and reservoir are preferably tested and optically examinedfor successful deployment. The liquid reservoir is preferably initiallytested to prove that it can be emptied of liquid contents. Initial testsare preferably also done on adsorbent surfaces. Further testing ispreferably done on a nonabsorbent surfaces to prove flow of liquid upthe capillary. Determination of the optimal flow rate for the reservoirand capillary combination is preferably determined based on glucoseconcentration at the patch detector.

The glucose detector patch material is preferably tested for sensitivityusing standard in vitro wet chemistry methods, to assure that itscellulose platform-glucose detector material is capable of reflectancedensitometric detection of at least 10 fg of glucose per μm².

In vitro (using cadaver skin) and in vivo animal and human biomechanicaltests of FDA approved biocompatible adhesives and adhesive membranesobtained from 3M, Inc and Adhesives Research, Inc, are preferablyconducted to determine optimal adhesive components and skin preparationconditions for occlusive, fluid tight adhesion requirements of the B-FITdevice. Upon completion of initial tests of the B-FIT system,preclinical dermatoxicological testing begins. These tests preferablyconsist of a demonstration of the biophysics of the device, done invitro using human cadaver skin or animal skin, and evaluation of localdermatological effects, done on live animals.

Dermatoxicological testing is preferably undertaken to demonstrate thebiophysical properties of the B-FIT device. Biophysical testing ispreferably conducted on animal or human cadaver skin. Full thicknesshuman abdominal skin specimens can be obtained commercially from Vitron,Inc. (Phoenix, Ariz.) and other vendors. Animal tissue samples canpreferably be used to establish a baseline and mitigate costs. The skinsamples preferably serve as a platform to investigate and optimize thethermal ablation mechanism. The B-FIT system has several different waysto ablate skin. The goal of the heat/ablation step is to remove thestratum corneum with no damage to the viable epidermis. The first set ofexperiments preferably determines the optimal ablation conditions, forexample, temperature peak, pulse duration, number of pulses, amongothers.

Tests to determine the optimum ablation conditions are preferablyaccomplished using optical and electron microscopy and surfaceprofilometry using an atomic force microscope in order to view andmeasure (a) depth and volume of ablation hole, and (b) the epidermalcell structural integrity so as to provide sufficient ablation of thestratum corneum without penetrating the viable epidermis.

To obtain a preclinical evaluation of safety, in vivo animal testing ofthe B-FIT system is preferably undertaken, utilizing, for example, ahairless rat, guinea pig, or fuzzy rat specie. Clinical observations forgross evidence of skin irritation, ulcer formation, and inflammatoryreactions are preferably made. Skin biopsies, examined using light andelectron microscopy provide a closer examination of the devicebiophysical effects. Serial clinical and microscopic observationsfollowing removal of the device enable assessment of the healing timefor the thermal ablation lesions.

Clinical pharmacological testing is preferably undertaken to determinethe analytical precision and accuracy of methods to determine glucoselevels via transdermal sampling relative to previously validated plasmaassays. A preferred assay technology is based on glucose oxidaseimmobilization in micromachined capillaries. A validated plasma assayfor glucose with acceptable limits of detection and quantification andwith acceptable intra- and inter-day coefficients of variation is usedto compare with these assays in the clinical settings described in thetwo trials outlined in detail below. The disclosed trials are ofidentical design: the first in normal volunteers, and the second inpatients with Type II (Adult Onset) diabetes mellitus.

In order to validate the analytical sensitivity of transdermal samplingto measure glucose, ten healthy men and non-pregnant women who havesigned an informed consent, fasted overnight, and have been screened tosatisfy the inclusion and exclusion criteria of the study are enrolledin a clinical trial to measure glucose concentrations in their plasma orinterstitial fluid before and during a glucose tolerance test. The B-FITsystem is attached to the dorsal surface of the right hand usingadhesive tape. An 18-gauge intravenous catheter is inserted in a forearmvein in the left arm. Venous blood samples (approximately 5 cc, and notless than 4 cc) are taken at appropriate intervals for the determinationof plasma glucose concentrations. Concentrations of glucose in theplasma are determined by a validated assay, routinely used in clinicalsettings. These concentrations are compared to those determined ininterstitial fluid using the B-FIT system. Plasma concentrations aremeasured on 8 occasions at 15 minute intervals over two hours, whileinterstitial fluid concentrations are measured for 0.5, 1, 2, 5, 10 and15 minute periods over the same 2 hours before the administration of 75grams of glucose by mouth. These data are used to optimize the samplingtime for the B-FIT system. After the administration of glucose, plasmaconcentrations and B-FIT system-estimated concentrations are measured at30 minute intervals for a further two hours. In healthy volunteers, theglucose plasma concentrations should range from 80 to 140 mg/dl underthese conditions (Washington Manual of Medical Therapeutics, 28thedition., 1995.) Inclusion criteria for the clinical trail are, asfollows: Group 1: men and women who are over the age of 21 years andunder the age of 75 years, Group 2: male and female volunteers who areover the age of 21 and under the age of 75, and who carry the diagnosisof adult-onset diabetes by a board-certified endocrinologist. Group 1:taking no prescription medications or natural products. Group 1: showingclinically normal laboratory values for complete blood counts, serumchemistries (Na, K, Cl, HCO₃, BUN, glucose and creatinine) andclinically normal liver enzyme profiles: SGOT, SGPT, alkalinephosphatase and bilirubin; ability to understand and carry out a signedinformed consent describing this protocol.

The following subjects are excluded from the trial: subjects who, in theopinion of the investigator, is noncompliant with the protocolrequirements; and women who are pregnant.

Once a subject has consented to participate in the study, the followingprocedures are conducted. Screening procedures are conducted within 21days of study initiation and include: medical history and physicalexamination; review of inclusion and exclusion criteria; and blood andurine specimen collection. Subsequent to inclusion in the study,subjects undergo the following procedures: (1) subjects arrive at thelocation of the clinical trial at approximately 9a.m. in the morningafter an overnight fast. Vital signs (heart rate, respiratory rate,blood pressure and temperature) are recorded.

The B-FIT system is placed on the dorsal surface of the right hand andattached securely with tape. Recording occurs via a 50 micron cauterizedlesion in the skin made by a small needle on the underside of themonitor that is not visible. The monitor is checked to ensure that it isrecording. Vital signs (heart rate, respiratory rate, blood pressure andtemperature) are recorded once the device has been attached once more.Samples of venous blood (5 cc or one teaspoon) are drawn from a catheterinserted in the a left forearm vein for the measurement of glucoseaccording to the above schedule while the subject is supine. Additionalblood samples are drawn four hours and eight hours after the first. TheB-FIT system monitor tape and device is removed. Patients are dischargedand allowed to return home.

With regard to the blood sampling schedule, five mL venous blood samplesare collected in vacutainers in the manner described above. The totalnumber of blood draws during the course of the study including thescreening samples is 14, (12 study draws and 2 screening draws forhematology, chemistry, and liver enzymes, respectively.) The totalvolume of blood drawn should not exceed 100 mL. Vital signs (heart rate,respiratory rate, blood pressure and temperature) are taken before andafter placement of the device and catheter and after the last blood drawhas been taken and the device and catheter has been removed. Patientsare encouraged to report any notable irritation on the arm where thedevice is placed. A physician is constantly available to subjectsenrolled in the study for concerns related to bruising or infection inthe skin due to the intravenous catheter or multiple blood draws. Inaddition, symptoms of polyuria and polydypsia are carefully noted andpaid attention to during the study with diabetic patients, and insulinis available for immediate injection by physicians and nurses should theneed arise. Statistical analysis include plasma glucose concentrationsdetermined using the clinical plasma assay as compared with the valuesobtained using the B-FIT system. If the correlation coefficient is >0.8with a significance p<0.05, the measurements are deemed valid.

The second clinical trial with diabetic patients is conducted using anidentical study design. Patients are allowed to take oral hypoglycemicmedications on the day before, but not on the morning of the study, andare asked not to inject insulin during the study period: Once the studyperiod is over, patients are allowed to eat and to resume their routinediabetic regime. In addition to the safety considerations describedabove, careful clinical monitoring and the availability of insulin ispaid great attention to while these subjects are under study.

The B-FIT system preferably utilizes many different microfabricationtechnologies and strategies, ranging from simple bulk micromachining tothe more complicated deep reactive ion etching (DRIE). Referring back toFIG. 5, a cross-section of a preferred one channel system microdevice isshown, preferably comprising of three main components: (1) the main bodycontaining several serpentine capillary channels, each with its ownreservoir channel, to sample and analyze physiologically compatiblefluid; (2) a bottom capping section to form the lower part of theserpentine structure and to contain micro-heating elements to thermallyporate the epidermal layer for substantial physiologically compatiblefluid extraction; and (3) a top capping section which forms the upperpart of the serpentine channel, and, if necessary, to contain electrodesfor assisting the flow of physiologically compatible fluids usingelectro-osmotic pumping through horizontal segments of the serpentinechannel. The first and second components together form the disposablemodules of the system. These interchangeable B-FIT elements are insertedinto the main connection receptacle after all analysis capillaries havebeen used.

The reservoir and capillary channels are preferably fabricated in astandard silicon wafer using deep reactive ion etching in order toobtain narrow, high aspect ratio through-wafer holes. The capillarychannels are preferably designed to be 25 μm in diameter with a nominallength of 500 μm, while the reservoir channels are 50 μm in diameter,but etched slightly less than 500 μm. The lateral portion of theserpentine capillary channel is preferably formed by recessing thesilicon surface by 25 μm. This region also preferably has a highlyreflective metal deposited on the surface to facilitate the mechanismfor optical detection of the analyte. After bonding the silicon with atop capping section, the serpentine structure becomes complete. Withthese dimensions, physiologically compatible fluids, such asperspiration or interstitial fluids, can be drawn into the open ends ofthe channels through capillary action. Furthermore, the fluid within thereservoir is preferably used in conjunction with capillary action, andwashes over the dermal region being tested, thereby assisting in thetransport of the physiologically compatible fluids through the smallerchannel. By activating the internal capillary channel surfaces tosustain a specific antibody immobilization, the fluids can be preferablyanalyzed by antibody-antigen complexation. A series of such capillaries,each with its own reservoir channel, is contained within a single deviceelement. Each analysis capillary and reservoir pair is preferablyaddressed individually to expose only one such pair to the skin surfacein order to perform a single fluid analysis. Once employed, the open endof the capillary continues to remain exposed to the skin.

The bottom capping unit is also preferably made using silicon and servestwo other major functions, aside from the role of forming the lowerstructure of the serpentine channel. Micromachined heating elementsincorporated within this section are preferably used to thermally poratethe skin surface, allowing greater availability of interstitial andphysiologically compatible fluids within the channels. Simultaneouslyoccurring during the stratum corneum poration procedure, themicro-systems are preferably used to individually address each of thecapillary-reservoir pairs. Initially, all open ends of the channels incontact with the skin are covered by a seal that can be “blown” toreveal a single analysis capillary. This cracking procedure can beeffectively controlled using large thermal gradients in close proximityto the seal, such as those afforded by silicon micro-resistors. Themicro-heaters are preferably integrated in the silicon regionsurrounding each of the capillary-reservoir channels. The connectingmicro-capillaries in this section are preferably formed using DRIE, eachbeing aligned with the vertical micro-capillaries from the main body.

Unlike the two previous components, the top capping layer is preferablymade out of plastic and used to accomplish several tasks. Firstly, itcompletes the upper structure, or lateral portion, of the serpentinechannel. This area, preferably, is the detection region of ourmicrosystem. Secondly, by using plastic, an imbedded waveguide canpreferably be fabricated within the material, with its orientationrunning parallel to the silicon surface and forming the basis of theintegrated photonics analysis system. In addition, to couple the lightfrom the waveguide into the detection region, a micro-mirror ispreferably integrated within the plastic. In addition, for moreeffective light coupling and better efficiency, a micro-lens ispreferably integrated within the plastic located directly above thedetection region. The micro-mirror is preferably integrated as a pressedcomponent within this top section by using a triangular form to indentthe plastic. The resulting indentation preferably has a 45° angle withrespect to the surface. Deposition of a highly reflective material ismade on the resulting beveled angle to render a micro-mirror thatreflects the horizontally-directed light from the waveguide downward.The integrated microlenses can also be stamped directly into the plasticor can be incorporated as separate units placed within the plastic byusing injection molding. In either case, the lens does not need to be ofhigh quality, but should simply be able to diverge light originatingfrom the waveguide. The illumination produced by the light from thewaveguide will cause the tagged analytes to fluoresce. The lightproduced is then reflected from the bottom surface of the detectionregion back through the now converging lens.

Returning to FIG. 6, the three possible (controllable) states of theindividual microcapillary systems are shown. The leftmostmicro-capillary system (#1) shows an exhausted capillary pair that hasalready been used for an analysis procedure. This first pair shows thecompleted thermal ablation, microfluid flow and capture of glucose fromexposed interstitial fluid, encounter with the glucose detection patch,and bluish color reaction evident at the upper surface of the chip. Themiddle micro-capillary system (#2) is performing an on-demand analysis.The rightmost capillary system is ready for a future, on-demandanalysis.

A purpose of the microdevice is to facilitate the transfer of moleculesof glucose or other poorly permeable analyte(s) from interstitial fluidin the viable epidermis, located just beneath the inner surface of thestratum corneum, to the detection patch situated on top of themicrodevice. The microdevice enables contact of the microfluidicsampling fluid directly with interstitial fluid by thermalmicro-ablation of the stratum corneum. By direct interface withinterstitial fluid, the microdevice enables sampling of, not only thenormally-inaccessible polar molecules, but also impermeable largermolecules such as proteins.

The first in a preferred programmed sequence of events is the flow ofelectrical current through the reservoir heating element to create aminute hydraulic pressure in the sealed reservoir containingphysiologically compatible fluid. The second and third steps occuralmost in unison, and comprise two separate currents through both thebreakable seal and the micro-ablation heater. The seal preferably is ametal-dielectric bilayer that ruptures at elevated temperatures. Themetal seal is preferably surface deposited on a low-stress silicondielectric element to reduce the chances of compromising the sealintegrity prior to its operation. Once the seal is broken during ananalysis procedure, the physiologically compatible fluid preferablyflows down from the reservoir and across the region that has beenthermally ablated by the micro-heater. During the time the seal is beingruptured, the micro-ablation heater is preferably being pulsed withalternating current to thermally remove successive layers of the stratumcorneum, which is typically about 30-60 μm in thickness. Themicro-ablation preferably occurs in a highly confined volume of thestratum corneum, approximately 50 μm×50 μm×30 μm. The physiologicallycompatible fluid from the now-open reservoir interfaces with theinterstitial fluid and, due to the dual actions of the reservoir heaterand capillary force, the mixture is transported towards the detectionpatch. The bulk of the physiological sampling fluid is preferably forcedout of the reservoir, emptying over the skin surface region and into theabsorbent detection patch. In addition, a strong, Band-Aid-like adhesivefilm preferably keeps the microdevice in fluid-tight contact with theskin, preventing escape of interstitial and physiologically compatiblefluids from the analysis region. The fluids are preferably forced up theanalysis capillary to the detection patch, directly above thecapillaries on the microdevice and, for example, in one embodiment,generate a color change to indicate the presence of glucose.

The microsystem component of the present invention is preferably basedon molecular scale manipulation using enhanced transdermal transfer ofmetabolites from interstitial fluids, and resultant detection withenzyme immobilized chemistries. Samples are preferably collected using aminimally non-invasive transdermal microdevice and trace quantities ofanalytes, which reach the skin surface by passive diffusion frominterstitial fluid underlying the outermost layer of skin (the stratumcorneum) can be detected. Since these analytes originate from otherparts of the body, transported to the interstitial fluid via bloodcirculation, they reflect a variety of physiological processes includingbody exposure to environmental chemicals or microbes, as well asinternal metabolism. Micro-layers of the stratum corneum are gentlyremoved enabling uptake of interstitial fluid from the viable epidermis,which lies just beneath the stratum corneum.

A preferred detection scheme for determination of health and otherimportant biological markers utilizes similar surface and biochemistryfor each assay. Returning to FIG. 8, a preferred procedure for thedetection scheme is shown. A preferred procedure is to covalently attachantibodies of the protein or metabolite of interest to a capillary wallthat incorporates a fluorescently tagged antigen. The tagged antigen isreplaced by competitive binding with the protein or metabolite ofinterest from the interstitial fluid sampled. The fluorescent antigen iskicked off into solution to be detected down stream in the collectingchamber by the photonics component. The fluorophore excitation andemission characteristics are matched to the photonics and visa versa forintegrating the right wavelength source, detector and filters toproperly excite and determine emission in the photonics module.

As an example, the following three proteins of various molecular weightscan be used for monitoring health properties: troponin I, C-reactiveprotein, and prealbumin. Anti-troponin I is covalently attached to thecapillary wall following a silane surface treatment ofaminopropyltrimethoxysilane (APTS). Troponin I is fluorescently taggedusing either fluorescein or rhodamine and bound to the antibody attachedin the capillary. Using competitive binding of troponin I from sampling,the fluorescently tagged troponin I is replaced into solution anddetected downstream. The above procedure can also used for bothC-reactive protein and prealbumin, albeit modified to take into accountdifferences between these proteins.

The surface chemistry is characterized stepwise to ensure sufficientsurface coating. The amount of bound antibody and competitive bindingstudies is tested using a variety of different instruments such as XPS,fluorescent plate reader, fluorescent microscope, or separationtechniques.

In another example, polyclonal antibodies raised against the caffeinemetabolites 5-acetylamino-6-formyl-3-methyl urea (AFMU) and1-methylxanthine (1×) are immobilized on the micromachined capillaries.The capillary tubes are modified by chemical treatment in order tointroduce hydroxyl moieties on the capillary surface. The surfacehydroxyl groups are then reacted with APTS, producing a molecular tetherwith free amine moieties at the end of the three-carbon chain.

The sugar residues in the Fc region of the antibodies raised againstAFMU and 1× are oxidized using periodate to generate aldehydes. Theantibodies are anchored to surface of the micromachined capillariesthrough Schiff base formation between the aldehydes on the antibodiesand the amines on the molecular tether. In this manner, the antibodybinding regions is directed away from the surface of the micromachinedchannels.

The amount of antibody immobilization on the surface of the microchannelis preferably determined by analysis of the protein content of thebinding solution before and after exposure to the microchannels. Thebinding activity of the immobilized antibodies is determined usingdisplacement of the fluorescent-labeled AFMU and 1× probes and theobserved activity is compared to the activity of equivalentconcentrations of non-immobilized antibodies to yield binding affinityper mg of immobilized antibody indices (BAI).

For the caffeine metabolites, in vivo and in vitro testing is conductedto assess the specificity of the antibodies AFMU and I X. The ability tomeasure the ratio of these metabolites using the device of the presentinvention is assessed by comparing the ratio obtained using the portablebiomedical monitoring system with the ratio obtained employingconventional HPLC methods. Devices modified with this assay are testedboth in vivo and in vitro for provide a preclinical evaluation. Thesedata are utilized as a baseline and preliminary data for testing thealgorithm.

Antibodies for prealbumin, CRP, troponin I are currently available onthe market and are used to assess the specificity of these antibodies totheir protein compliment. As some commercial antibodies are not activeor specific, this prescreening test is preferred to determine activityand specificity for each protein of interest. Specificity is preferablytested for each antibody by adding other substances similar instructure, which should not cross-react. For example, in assessingprealbumin the proteins such as albumin and globulins, among others, areadded. In assessing caffeine metabolites, xanthines and xanthinemetabolites are added. Assays using the antibody, the ligand and thefluorescently labeled ligand are preferably developed using techniquessuch as flow injection analysis (FIA).

Each completed assay is assessed for accuracy, reproducibility,linearity, and results are compared with those of existing procedurescurrently used in the clinical laboratory. A preclinical evaluation forin vivo and in vitro testing is done and the data is utilized in thealgorithm developed.

The portable biomedical monitoring system of the present invention ispreferably based on molecular scale manipulation using enhancedtransdermal transfer of metabolites and other body analytes usingtransdermal dosimetry immobilized antibodies in microchannels, capillaryaction for fluidic mobility, and integrated photonics for detection.Thus, the micro-fluidic chip interface technologies of the presentinvention provide controlled sample collection from host fluids,(circulatory and noncirculatory) and for the controlled delivery offluids (drugs, chemicals) and target probes (antibodies, proteins,signal molecules). In addition, sample collection platforms of thepresent invention can simultaneously employ an “outward facing” devicecomponent for sampling air or liquid borne environmental target analytesand an “inward facing” component for detection of target analytesemanating from the skin surface or accessible body fluid.

The apparatus and process of portable biomedical monitoring disclosedherein is adaptable to a wide variety of chemistries. For example, theportable biomedical monitoring device can include chips which monitorhealth (Chip “A”) and illness or infection (Chip “B”). Chip A canmeasure molecules like glucose to establish a baseline of the subject'shealth state in both normal and high stress situations. Changes fromthese baseline limits will signal a need for Chip B. Chip B is designedto determine the exact cause of illness. For example, Chip B can containantibody conjugates for parathion and its metabolites that emulate achemical warfare agent. The structure of the microsystem is adaptablefor many other types of chemistries based upon drag metabolism and/or“probe drugs”.

Drug metabolism is the process by which drugs are converted, byenzyme-catalyzed reactions, to products or metabolites which are readilyexcreted in the urine and bile. One pathway of drug metabolism are phaseI reactions, which involve the creation or modification of a functionalgroup in the substrate molecule. The cytochrome P450-dependent (CYP)microsomal mixed function oxidase system is a very important enzymesystem for these reactions. A second major pathway involves phase IIreactions, in which the drug or a phase I metabolite is conjugated witha water soluble endogenous substrate. Phase II reactions involve adiverse group of enzymes known collectively as transferases. This groupincludes UDP-glucuronyltransferase, UDPglycosyltransferase,glutathione-S-transferase, sulphotransferase, methyltransferase, andN-acetyltransferase.

Drug metabolism is affected by dietary and environmental factors. Forexample, alcohol, certain food constituents and compounds in cigarettesmoke have been observed to affect the biotransformation of many drugs,as have industrial pollutants and pesticides. Genetic factors also playan important part in the control of drug metabolism and it has beenobserved that there is much variation in drug effects betweenindividuals. For some enzymes, discrete genetic subgroups are present inthe human population. These genetic polymorphisms are generated bymutations in the genes coding for these enzymes which cause decreased,increased or absent enzyme expression or activity. Genetic polymorphismsof several CYPs have been identified and their activity falls into twoclearly defined and qualitatively different populations: individualswhose rate and extent of metabolism is poor (poor metabolizers, PMs) andthose who have faster or more extensive metabolism (extensivemetabolizers, EMs). Genetic polymorphisms of some phase II enzymes alsoexist. For example, N-acetyltransferase-2 (NAT-2) is affected in thisway and this acetylation polymorphism relates to the metabolism of avariety of drugs and carcinogens. Numerous alleles are associated withdecreased function of this enzyme and a bimodal distribution isobserved: 50-60% of individuals are genotypically slow acetylators andthe rest of the population are fast.

In healthy individuals, the metabolic genotype normally predicts themetabolic phenotype. That is, for a particular enzyme, genotypicallyextensive metabolizers are observed to efficiently metabolize drugs thatare substrates for that enzyme, and genotypically poor metabolizers aredeficient in that process. However, drug interactions, infection,disease progression and malnutrition may produce changes in the relativelevels and activities of metabolizing enzymes. Thus, in healthyindividuals the relationship between genotype and its expression(phenotype) is conserved; i.e. FAST genotypes produce FAST phenotypes,while SLOW genotypes produce SLOW phenotypes. However, a disease stateof the individual can alter this relationship, as can diet, smoking,alcohol, environmental chemicals, and biological or chemical warfareagents. For this reason, the determination of metabolic phenotype (themeasure of actual enzyme activity) is of great importance and can beused as a direct and sensitive probe of health and clinical status. In apreferred embodiment, identification and quantification of specificmetabolite patterns produced by innocuous test compounds or probe drugscan be utilized to determine the metabolic phenotype of a subject. Forexample, caffeine is metabolized by several routes including oneinvolving NAT-2. Thus the urinary ratio of 2 metabolites,5-acetylamino-6-formylamino-3methyluracil (AFMU) to 1-methylxanthine(1×) is an index of NAT-2 activity.

Examples of numerous embodiments follow. Each embodiment can bepracticed alone or in conjunction with other embodiments of theinvention.

For example, as mentioned above, dispositional or metabolic markers of“stress” can be monitored, including but not limited to, chemistries forthe detection of different chemical probes of human health, such asglucose, caffeine, ethanol, and dextromethorphan. “Stress” can manifestitself via detectable alterations of many internal metabolic pathways,such as in altered insulin-glucose patterns or aberrant hepaticcatabolism of safe, commonly used stimulants (caffeine) orantihistamines (dextromethorphan).

The enzyme N-acetyl transferase (NAT-2) metabolizes caffeine. Thisenzyme is highly polymorphic. The activity of NAT-2 is known to beassociated with adverse drug effects, diverse toxicities andpredisposition to disease. Two major metabolic phenotypes have beenidentified: fast and slow N-acetylators. The expressed activity of NAT-2(phenotype) has been shown to be affected by acute and chronic diseasestates. For example, in HIV+ and AIDS patients, the presence of an acuteillness reduces the expressed activity of NAT-2, changing a patient witha fast NAT-2 phenotype into one with a slow NAT-2 phenotype. When theillness is resolved and the patient is returned to the initial clinicalstate, the patient again expresses a fast NAT-2 phenotype. Thus, thedetermination of an individual's NAT-2 phenotype and the monitoring ofchanges in this phenotype can be a direct and sensitive probe of thatindividual's health and clinical status. This determination allowsprediction of whether patients are FAST or SLOW metabolizers prior toinitiating drug regimens. This approach also allows for the screening ofall patients before drug treatment is initiated so that appropriatedosage regimens are given at the outset of treatment and drugovertreatment or undertreatment is avoided.

The NAT-2 phenotype can be determined by a number of probes. In thepreferred embodiment, caffeine is used because of its wide distributionand relative safety. In studies using caffeine as the probe, thephenotype of the enzyme is determined by the ratio of two caffeinemetabolites: AFMU to 1X. Based on the ratio of these metabolites, theactivity of the enzyme can be determined. Polyclonal antibodies aregrown against the metabolites AFMU and 1× and then purified. Theseantibodies are successfully used to determine NAT-2 phenotypes.

The preferred detection scheme consists of the anchoring of antibodiesof a particular metabolite or chemical antigen to the surface of thecapillary. The antibody is bound to a special antigen attached to afluorescent tag such as rhodamine. As the antigen flows into the channelit will release the fluorescent tag which is detected downstream.

Thus, in an alternative embodiment, phenotyping using NAT-2 is conductedto indicate an infected or diseased state. The enzyme NAT-2 is highlypolymorphic. The activity of NAT-2 has been associated with adverse drugeffects, diverse toxicities and predisposition of disease. Themonitoring of changes in this phenotype is a direct and sensitive probeof the soldier's health and infection status.

In a further embodiment, organophosphate chemicals (nerve) agents aremonitored using the insecticide surrogate model compound parathion.Since “nerve gas” type chemical weapons like Tabun, Sarin, and Soman actby inhibition of acetylcholinesterase, the organophophate insecticide,parathion (or its metabolites) provides an excellent “surrogate” analyteto detect exposure. A parathion monitor, thus, has important industrialand civilian applications.

In a further embodiment, inflammatory sequeli to microbial toxins aremonitored, including, for example, interleukin-1 (IL 1); interleukin-6(IL 6); and tumor necrosis factor (TNF); among others. Circulating IL 1,IL6, and TNF present candidate analytes that can be collected anddetected using permeation enhanced transdermal techniques and advanceddetection system designs in accordance with the present invention.

In a further embodiment, microbial toxins are monitored, including, forexample, anthrax, botulinum toxin, endotoxin, among others. Althoughmicrobial toxins are typically large molecules, their extremely highbiological potency, coupled with enhanced outward migration usingmicroscopic physical barrier modification techniques (for example,thermal microablation) can permit transdermal dosimetry employingdetection systems incorporating toxin responsive components. Inaddition, antibody tags can be used for identification of infectingagents and determination of bacterial and viral loads. Moreover, thedetermination of D-amino acids (from bacterial sources) can enable themonitoring of the response to antibiotic therapy.

In a further embodiment, spore metabolites can be monitored. Circulatingbiochemical metabolites arising from human catabolism via lymphatic andhepatic pathways of microbial spores are collected and detected usingthe techniques and optimized detection system designs of the presentinvention.

In a further embodiment, specific proteins are monitored, such as thosereferred to in the table below. Concentration Mol. Wt. Protein (mg/L)(kD) Prealbumin  70-390 54 C-reactive 0.06-8.2  115-140 Protein TroponinI <0.0001 76

Prealbumin (MW 54,000) is known as being an important marker fornutritional status. The reference range for 0-1 month old is 70-390mg/L. Uses of this embodiment include but are not limited to screeninginner city pediatric populations for nutritional status, as well asscreening all patients for nutritional status, particularly prior tosurgery.

C-reactive protein (MW 115,000-140,000) is an acute phase reactant andas such is elevated in many disease processes. The reference range inadults is 68-8200 μg/L. The measurement of this protein provides a goodindication of health vs. disease. C-reactive protein is also animportant prognosticator of heart disease and impending myocardialinfarction. Thus, this assay could also be used to screen forcardiovascular health.

Troponin I is recognized as a useful and specific marker for acutemyocardial infarction. The reference range in adults is <0.1 μg/L. Inmyocardial infarction patients it is >0.8 μg/L This assay provides areal time evaluation of troponin I in the emergency rooms of hospitalsand provide the earliest recognition that a patient needs to be admittedto intensive care units.

In addition to monitoring, the biomedical monitoring system of thepresent invention can provide drug delivery with feedback control inbursts to maintain concentrations of a specific agent within the body atspecific levels throughout the day, levels which can vary on a day today basis and during the day. Examples of such agents include thehormones estrogen and testosterone. The decrease that occurs in estrogenwith age is intimately related to the increased risk of osteoporosis andcardiovascular disease in women. Moreover, the replacement withpharmacologic estrogen may improve mortality from cardiovasculardisease, reduce the risk of osteoporotic fractures and may play animportant role in protecting women against Alzheimer's disease. Thesediseases have immense societal impact and financial cost, but theirtreatment with replacement estrogen is associated with a host of sideeffects including, not least the development of breast cancer anduterine cancer, but also a host of other effects including skin changes,weight changes and depression. Although no medicine has been shown to beas effective as estrogen itself, a huge effort has been expended todevelop modified estrogens that have selective actions on bone, breastor other tissues (the development of specific estrogen receptormodulators or SERMs). The approach of administering effective estrogenin a physiologic, controlled and monitored manner is attractive in thatit remains the most effective medication and innovative therapeuticregimens utilizing it may prove of great benefit.

The delivery of testosterone in a controlled and monitored manner canalso be useful. Serum concentrations of testosterone also decline withage, as they do in a number of pathological conditions, including HIV.Testosterone replacement strategies for the treatment of HIV and cancerwasting, male osteoporosis and chronic obstructive pulmonary disease areemerging and can also be useful for short term controlled administrationpost-operatively after major surgery to enhance the rate and thelikelihood of successful recovery. Feasibility of these embodiments isinvestigated through transdermal detection of estradiol (E2) in Rhesusmonkeys and human females. A prototype solid phase E2 detection system(TED) can be incorporated in a transdermal patch that immobilizesantibody against E2 in the TED, and analyzed ex-situ using aradioimmunoassay procedure. The E2 detection system is capable ofdetection less than 0.125 picograms. TEDs are first tested byemplacement for 24 hours on the chests of partially or fully castratedfemale Rhesus monkeys (n=3), treated with placebo or 20 ug/kg estradiolbenzoate. The TED measurements distinguish between monkeys that havehigh circulating E2 concentrations and those who have none. TEDs canalso be affixed on the forearms of four reproductive age human femaleswho exhibit a large range of circulating E2 concentrations (48-382pg/ml). E2 collected in TEDs range from 0.06 to 0.5 pg, and correlateroughly with circulating E2 concentrations. These data are consistentwith an in vivo permeability coefficient of 4.3±0.5×10′5 cm/hr.

In a further embodiment, the portable biomedical monitoring device ofthe present invention can be used for pain management, determining howbest to deliver codeine and morphine, among others, to minimizecytotoxicity, while achieving pain control.

In a further embodiment of the present invention, a MEMS-basedphysiochip can be used to non-invasively monitor fundamentalphysiological aspects related to human function under typical andatypical environmental conditions. By carefully monitoring of relevantphysiological data such as body temperature, pulse rate, blood pressure,and heart activity (electrocardiogram) an infinitesimal change oranomalous behavior can provide an early indicator of stress to the humansystem.

In a further embodiment, passive or non-invasive transdermal dosimetryis used without physical or chemical modification of the normal skinbarrier. This embodiment is practical for small molecular weightanalytes that exhibit both lipid and water solubilities.

The following description of experiments and clinical trials is providedso as to demonstrate how various embodiments of the present inventionperform. Suitable analytes for demonstrating the operation of theseembodiments of the present invention are provided below. However, it isto be recognized that the systems and methods of the present inventioncontemplate analysis of a much larger set of analytes in the variousembodiments of the present invention.

Development of immobilized nicotinic acetylcholine receptor (nAChR)based-HPLC stationary phases and the application of these phases to theon-line determination of drug-receptor interactions.

Preparation of nAChR-detergent solution. Rat whole forebrain ortransfected cells are suspended in 50 mM Tris-HCl, pH 7.4, (buffer A),homogenized for 30 seconds with Brinkmann Polytron, and centrifuged at40,000×g for 10 min at 4° C. The pellet is resuspended in 6 ml of 2%deoxycholate or 2% cholate in buffer A and stirred for 2 hours. Themixture is centrifuged at 35,000×g for 30 minutes, and the supernatantcontaining nAChR-deoxycholate solution is collected.

Immobilization of nAChRs on MMparticles or Superdex 200 gel beads. DriedIAM particles are suspended in 4 ml of the obtained detergent solutionscontaining nAChR subunits or subtypes. For the immobilization of onenAChR subtype, the mixture of IAM-detergent-receptor is stirred for 1hour at room temperature. The suspension is dialyzed against 2×1 Lbuffer A for 24 hours at 4° C. The IAM LC support with immobilizednAChRs is then washed with buffer A, centrifuged and the solidcollected.

A dried lipid mixture of 60 mg L-α-Lecitin (20% phosphatidylcholine), 10mg L-α-phosphatidylserine, and 20 mg cholesterol is solubilized with 4ml of obtained nAChR-detergent solution. The nAChR-lipid-cholatesolution is mixed with 50 mg dry Superdex 200 beads. The suspension isdialyzed against buffer A for 24 hours at 4° C. Non-immobilizedliposomes are removed by centrifugal washing with buffer A at 2,000×g.

([³H]-epibatidine ([³H]-EB) binding assays for the suspensions ofnAChR-IAM particles and nAChR-Superdex 200 beads: The nAChR-IAMparticles, IAM particles, nAChR-Superdex 200 gel beads and Superdex 200gel beads, corresponding to 30 mg dry material, are each suspended in1.25 ml buffer A. A 250 gl aliquot of each suspension is incubated with250 gl of [³H]-EB [1.5 nM]for 4h at 24° C. in a final volume of 2.5 ml.

Experiments are carried out with and without added 100 μl of 300 μM(−)-nicotine. Bound and free ligands are separated by vacuum filtrationthrough Whitman GF/C filters treated with 0.5% polyethylenimine. Thefilter-retained radioactivity is determined by liquid scintillationcounting. Specific binding is defined as the difference between totalbinding and nonspecific binding. The amount of protein is determinedusing BCA reagent (Pierce, Rockford, Ill., USA) measured at 570 nm.

Chromatography based on nAChR-LAMcolumn or nAChR-liposome-Superdex 200column: The nAChR-IAM particles or nAChR-Superdex gel beads are packedin a HR5/2 glass column and connected to a HPLC pump. [³H]-EB is used asa marker and an on-line flow scintillation detector (525 TR) monitorsthe elution profile. All chromatographic experiments are performed atflow rate 0.4 ml/min at room temperature.

In zonal chromatographic experiments, a 100 μl-loop is used to apply thesample. The chromatographic data is summed up in 0.5-min intervals andsmoothed using the Microsoft Excel program with a 5 point movingaverage.

In frontal chromatogram 50-ml sample superloop are used to apply aseries of [³H]-EB concentration through the nAChR-column to obtainelution profiles showing a front and plateau regions. Thechromatographic data is summed up in 1-min intervals and smoothed usingthe Microsoft Excel program with a 10 point moving average.

Results: Immobilization of nAChR subunits or subtypes. About 63 mgprotein isolated from the membrane of transfected cells and 14 mg ofprotein prepared from the brain tissues are respectively immobilized onthe per gram of IAM particles or Superdex 200 gel beads. Receptorbinding assays using [³H]-EB showed that the nAChR binding activitiesare retained after the immobilization procedure as shown in the tablebelow. In parallel experiments, no specific binding of [³H]-EB isdetected on IAM particles and Superdex 200 gel beads. Specific nAChRDensity Sample Binding (%) (nmol/g protein) α4/β2 nAChR-detergentsolution 62 0.14 α4/β2 nAChR-IAM¹ 49 0.81 α3/β4 nAChR-detergentsolution² 100 8.57 α3/β4 nAChR-IAM2 97.8 5.09 α3/β4 nAChR-liposomeSuperdex 200² 29.4 1.45¹prepared from rat forebrain with detergent deoxycholate.²prepared from transfected cells with detergent cholate.

Frontal chromatography with α3/β4 nAChR-IAM stationary phase: Theretention volumes of [³H]-EB are 23 ml at the concentration of 60 pM.This retardation is primarily due to the specific binding to saturablesites of the receptors as indicated by a decrease in retention volume to8 ml when the concentration of [³H]-EB is increased to 450 pM (FIG. X,profile B). The binding of [³H]-EB to the α3/β4 nAChR-IAM stationaryphase could be reduced in competitive displacement experiments usingknown α3/β4 nAChR ligands in the mobile phase. For example, theretention volume of 60 pM [³H]-EB decreased from 23 ml to 18 ml when a60 nM concentration of the nAChR-ligand (−)-nicotine is added to themobile phase and fell to 0.9 ml when the (−)-nicotine concentration isincreased to 1000 nM. The decreases in retention volumes of [³H]-EBrelative to mobile phase concentrations of a displacer reflect thebinding affinity of the displacer for the receptor. Using thistechnique, the relative affinities of nicotinic drugs for the α3/β4nAChR are readily the relative affinities of nicotinic drugs for theα3/β4 nAChR are readily classified by determining the concentrationsrequired to decrease the retention volumes of [³H]-EB to a predeterminedlevel.

To decrease the retention volumes of 60 pM [³H]-EB from 9.5 ml to 6 mlon an α3/β4 nAChR column (0.5×1.25 cm), requires mobile phaseconcentrations of 0.12 nM of (±)-EB, 1.7 nM of A85380, 45 nM of(−)-nicotine, 1,200 nM of carbachol or 21,000 nM of atropine,respectively. The relative affinities of these drugs for the a3/(34nAChR determined by this method are therefore(±)-EB>A85380>(−)-nicotine>carbachol>atropine which is consistent withresults from ligand binding assays using membrane homogenates. Therelative affinities can be classified by the association constantscalculated from the resulting data in the table below. Ligand K_(d) ¹(nM) K_(d) ² (nM) (±)-Epibatidine  0.27 ± 0.05 0.38 ± 0.07 A85380  17.2± 0.5 73.6 ± 6.3  (−)-Nicotine    88 ± 33 475 ± 52  Carbachol  1,280 ± t30 3,839 ± 276   Atropine 14,570 ± 2600 —¹Frontal chromatography with a3/(34-IAM stationary phase (0.5 × 1.3 cm).²Binding assay using cell membrane homogenates.

These dissociation constants (K_(d)) values show the same rank order asthose of the values measured with binding assays using membranehomogenates. The low affinity of atropine (K_(d): 17,200 aM) is alsoconsistent with literature values.

Zonal chromatography for determination of different specific bindingactivities of immobilized nAChRs subtypes: Binding of [³H]-EB is alsomeasured in zonal format on the columns containing α3 subunits only, β4subunits only, a mixture of the two cell types, or α3/β4 nAChRs. Theretention of [³H]-EB on α3 nAChR-IAM (peak 1, FIG. 23 a), β4 nAChR-IAM(peak 2, FIG. 23A) and α3/β4 nAChR-IAM (peak 3, FIG. 23A) is low, and nosignificant change in the retention volumes is observed when adisplacer, (−)-nicotine, is included in the mobile phase, [³H]-EB isretained on the IAM column containing the immobilized α3/β4 nAChR-IAM(peak 4, FIG. 23A). The retention volume is decreased when theconcentration of [³H]-EB is increased or when (−)-nicotine is includedin the mobile phase, peak 4 (dash line) FIG. 23B.

Specific binding activities of immobilized nAChRs subtypes. The resultsof binding to immobilized receptors showed that [³H]-EB and (−)-nicotinehave higher binding affinities at nAChR α4/β2 subtype than atα3/β4-subtype and these results are consistent with the resultsdetermined from ligand binding assays using membrane homogenates asshown in the table below. The K_(d) values obtained from α4/β2nAChR-liposome-Superdex 200 column are similar as those determined usingα4/β2 nAChR-IAM column. K_(d) of K_(d) of Formats of nAChRs(±)-epibatidine (nM) (−)-nicotine (nM) α3/β4 - nAChR-IAM 0.27 ± 0.05 88± 33 α3/β4 - nAChR membrane 0.38 ± 0.07 475 ± 52  α4/β2 - nAChR-IAMB0.044 ± 0.005 1.0 ± 2.3 α4/β2 - nAChR membrane 0.053 ± 0.002 7.2 ± 1.3α4/β2 - nAChR-liposome- 0.020 ± 0.08  7.4 ± 2   Superdex 200

Effects of ionic strength and pH of the mobile phase on the binding of[³H]-EB: The effect of mobile phase ionic strength and pH on the bindingaffinities of [³H]-EB are determined with a α3/β4 nAChR-column. Theretention volumes increased when the pH of mobile phase is increasedfrom pH 4.0 to pH 7.0 and remained constant between pH 7.0 to 9.5. Theretention volumes of [³H]-EB are higher at low ionic strength (5-mMammonium acetate) and decrease as the ionic concentration of the mobilephase increases.

Stability and reproducibility of nAChR columns: One α3/β4 nAChR-IAMcolumn is used continuously over a ten day period and then stored for 40days at 4° C. The retention volumes for 60 pM [³H]-EB are 9.5±0.05 ml(from day 1 to day 10) and 9.7±0.08 ml (day 50). The relative affinitiesof EB and (−) nicotine obtained on three α3/β4 nAChR-IAM columnsprepared from different batch of cell lines are reproducible as shown inthe table below, although the retention volumes of EB at the sameconcentration differed from column to column. Column size K_(d) of EBK_(d) of (−)-Nicotine Binding sites (cm) (nM) (nM) (pmol/ml bed) 0.5 ×1.8 0.34 ± 0.04 52 ± 10  7.5 ± 0.2 0.5 × 1.3 0.27 ± 0.05 88 ± 33 13.5 ±0.3 0.5 × 1.7 0.21 ± 0.06 130 ± 45  15.0 ± 0.4

Preparation of Immobilized GABAA and nicotinic acetylcholine receptorson an IAM support from rat whole brain: Rat whole brain (4 brains) ishomogenized in 30 ml of TRIS-HCl buffer [50 mM, pH 7.4] containing 5 mMEDTA, 3 mM benzamidine and 0.2 mM PMSF (Phenyl methyl sulfonyl chloride)for 3×20 seconds using a Brinkman Polytron at setting 6. The mixture iskept in an ice bath for 20 seconds between each homogenization step toprevent excessive heating of the tissue. Homogenized brain tissue iscentrifuged for 10 min/4° C. at 21,000 rpm. Supernatant is removed usinga Pasteur pipette and discarded. The pellets are suspended in 10 ml ofSolubilization Buffer containing 100 mM NaCl, 2 mM MgCl₂, 3 mm CaCl₂, 5mM KCl, 2% Na-cholate and 10 μg/ml Leupeptin in TRIS-HCl buffer [50 mM,pH 7.4]. The resulting mixture is stirred for 12h/4° C. and centrifugedat 21,000 rpm.

Supernatant (receptor-cholate suspension) is mixed with 200 mg of driedIAM-PC packing material and stirred gently for 1 h/25° C., transferredinto dialysis tubing and dialyzed for 48 h/4° C. against 3×600 ml ofDialysis Buffer containing 5 mM EDTA, 100 mM NaCl, 0.1 mM CaCl₂ and 0.1mM PMSF in TRIS-HCl buffer [50 mM, pH 7.4].

The receptor-IAM-PC is centrifuged for 3 min/4° C. at 2,000 rpm.Supernatant is discarded. Pellets are washed with TRIS-HCl buffer [50mM, pH 7.4] and centrifuged until the supernatant is clear. Theresulting pellets are used to pack the column.

Determination of binding affinities to the immobilized GABAA receptor(GR) using frontal chromatography: The GR-IAM particles are packed in aHR5/2 glass column and connected to a HPLC pump. [³H]-Flunitrazepam([³H]-FTZ), a GABAA receptor ligand, is used as a marker and an on-lineflow scintillation detector (525 TR) monitored the elution profile. Allchromatographic experiments are performed at flow rate 0.4 ml/min atroom temperature. In frontal chromatography, a 50-ml sample superloop isused to apply a series of [³H]-FTZ concentrations through the GR-columnto obtain elution profiles showing a front and plateau regions. Thechromatographic data is summed up in 1-min intervals and smoothed usingthe Microsoft Excel program with a 10 point moving average.

When the GABAA receptor ligand diazepam (DAZ) is added to the mobilephase, the retention volume of [³H]-FTZ is reduced in proportion to theconcentration of DAZ in the mobile phase. These results indicate thatthe retention of FTZ on the GR-IAM is due to specific interactions withthe immobilized GABAA receptor. The dissociation constants (K_(d)) ofFTZ and DAZ are determined on the GR-IAM. The calculated K_(d) of FTZand DAZ obtained by frontal chromatography are consistent with thosedetermined by classical binding assays, as shown in the table below.Ligand Frontal Chromatography Binding Assays Flunitrazepam 1.3 1.7Diazepam 1.0 1.3Production and purification of the ER-LBD. The Estrogen Receptor (ER) ispart of the Nuclear Receptor Superfamily. It is made up of fivedifferent regions: A, B, C, D and E. The E region, also known as, theligand binding domain (LBD) is where the agonists and antagonists bind.The ER-LBD has been expressed in yeast and also in bacteria via a fusionproduct between protein A and the LBD. The Production of recombinantEstrogen Receptor Protein is described: The DNA sequence coding for theligand binding domain of the human estrogen receptor a protein (aminoacids 302-595) is obtained by PCR using the full length cDNA as thetemplate. The product of the PCR reaction is subcloned into the pRSETplasmid in frame with a 6 histidine tag on the N-terminal end of theprotein. The His tag is used for the purification of the protein fromthe bacterial proteins. The plasmid is transformed into the BL21 codon+bacteria. The bacteria are grown in standard LB Broth to an opticaldensity at X=600 of ˜1.5.

The bacteria are harvested by centrifugation and frozen at −80° C. untilfurther purification. The bacteria pellets are lysed in a urea/HEPESlysis buffer by sonication and clarified by centrifugation andfiltration. The lysate is loaded onto a 5 ml Ni—NTA nickel affinitycolumn that is preequilibrated with the urea/HEPES lysis buffer. TheNi—NTA column selectively binds proteins with the 6-His tag. Thenontagged proteins are washed off the column with the urea/HEPES buffer.The estrogen receptor is refolded on the column by gradually changingthe buffer to a PBS (phosphate buffered saline) buffer. Finally, theestrogen receptor protein is eluted with a PBS buffer containingimidazole, which competes with the His tag for binding to the Ni—NTAcolumn. The fractions containing the estrogen receptor protein aredetermined by gel electrophoresis and staining with Gelcode Blue and bywestern blot analysis using a antibody against the human estrogenreceptor. The concentration of protein purified is determined viabicinchoninic acid (BAC) protein assay.

Binding activity of the ER-LBD. A binding assay is carried out todetermine the activity of the fusion protein. The classical method usingdextran-coated charcoal is initially used and gives the activity of theprotein. However, the method is improved with the use of Nickel-NTAagarose beads to isolate the fusion protein. Roughly 200 pmoles ofprotein is placed per tube. For total binding, varying concentrations of[³H]-estradiol is added and for nonspecific binding a 200 fold excess ofthe cold estradiol is added prior to the addition of the radiolabeledestradiol. The solutions are incubated at room temperature for 2 hours.Following incubation, the Nickel-NTA is added. After one wash, theprotein is displaced with imidazole. The K_(d) is determined to beapproximately 3.4 nM (an average K_(d) of several experiments). Althoughestradiol had a slightly stronger affinity for the native ER (0.2 nM),this is sufficient.

Immobilization of the ER-LBD. The initial immobilization of the isolatedfusion protein is carried out using a silica based immobilizedartificial membrane: IAM.PC. This membrane contains a silica core, whichis attached to a hydrophobic spacer with a polar head group. Theprocedure for immobilization of the protein onto these membranes isknown in the art. Varying concentrations of IAM are used to determinethe optimal conditions for immobilization. It is determined that 25 mgof IAM is optimal with 35% incorporation.

However, upon testing for activity it becomes apparent that[³H]-estradiol is not only binding to the protein but also to thehydrophobic layer of the membrane. Increasing the ethanol concentrationin solution does not significantly reduce the binding to the membrane.Using a modified IAM stationary phase, the IAM-MG, that is morehydrophilic only slightly reduces the nonspecific binding.

The ER-LBD is then immobilized in a new column format containing asilica backbone and a hydrophobic spacer (C10). The ER-LBD isimmobilized and retained its binding activity but the nonspecificbinding of [³H]-estradiol is still to great for effective use of thecolumn. The C10 spacer is replaced by a hydrophilic spacer and thenonspecific binding of [³H]-estradiol is eliminated and the ER-LBD-SPcolumn is synthesized.

The K_(d) of the estradiol marker ligand is then determined on-lineusing the ER-LBD-SP column. The ER-LBD-SP column is connected withon-line flow scintillation monitoring (kadiometric FLO-ONE Beta 500 TRinstrument, Packard Instrument Co., Meridien, Conn.) and run at roomtemperature for 97.5 minutes at a flow rate of 0.2 mL/min. The systemsetup is as described by Zhang, et al., Immobilized Nicotinic ReceptorStationary Phase For On-Line Liquid Chromatographic Determination ofDrug-Receptor Affinities, Anal. Biochem. 264, 22 (1998). 18 mL samplesof 0.5 nM [³H]-Estradiol ([³H]-E2) supplemented with a range ofconcentration of cold Estradiol (0-7 nM) are run by frontalchromatography. The elution volume data is used to calculate thedissociation constant of the ligand. The K_(d) value of estradiol iscalculated by nonlinear regression with Prism (GraphPad Software) usingone site binding equation: Y=Bmax [E2]total/(K_(d)+[E2]total). The K_(d)values of estradiol is calculated as previously described to be(0.189±0.06) nM. The radioactive signal is recorded every 6 seconds byan on-line flow scintillation detector.

Preparation of the ER-LBD: The recombinant ER-LBD is obtained andpurified as described above.

Immobilization of the ER-LBD: The ER-LBD is then immobilized in themicromachined capillaries. The immobilization is accomplished throughactivation of the silanol groups on the silica chips usingdicyclohexylcarbodiimide (DCC) and then coupling of the C2 spacer with afree carboxyl group to the activated surface. The ER-LBD is then boundto the derivatized surface using the procedures developed in theprevious studies with the liquid chromatographic stationary phasecomposed of silica gel beads. The amount of protein immobilized on thesurface of the microchannels is determined by analysis of the proteincontent of the binding solution before and after exposure to themicrochannels.

If the initial experimental approach to the immobilization of ER-LBD isnot successful the following procedures are investigated: 1) if theproblem exists at the during the activation of the silanol groups at thesilica surface, DCC is replaced by dimethylaminopyridine (DMAP); 2) ifthe problem arises from the C2 spacer, C3 to C4 spacers are examined; 3)if a problem exists with the immobilization to the new surface, anepoxide activated approach is explored by a method such as described inJ. B. Wheatley et al.: Salt-induced immobilization of affinity ligandsonto epoxide-activated supports, J. Chromatogr. A, 849, 1 (1999); D.Zhou, et al.: Membrane affinity chromatography for analysis andpurification of biopolymers. Chromatographia, 50, 27 (1999), or anapproach utilizing streptavidin-biotinylation such as described by L. A.Paige, et al.: Estrogen receptor (ER) modulators each induce distinctconformational changes in ERα and ERβ. Proc. Nat. Acad. Sci., 96, 3999(1999).

Binding activity of the immobilized ER-LBD: The binding activity of theimmobilized ER-LBD is determined using [³H]-estradiol (0.005 nM inphosphate buffer [0.1 M, pH 7.4) ([³H]-E2) supplemented with a range ofconcentration of cold estradiol to produce a range of from 0.001 to0.050 nM in phosphate buffer [0.1 M, pH 7.4]). The solutions containingthe [³H]E2 are applied to the microchannels containing the immobilizedER-LBD, microchannels containing the immobilized support (without theER-LBD, a positive control) and bare microchannels (negative control).The solution containing microchannels is incubated at room temperaturefor 30 minutes. The channels are then washed three times with phosphatebuffer [0.1 M, pH 7.4], the washing is collected and assayed for [³H]-E2content using a scintillation detector. The K_(d) value of E2 iscalculated by nonlinear regression with Prism (GraphPad Software) usingone site binding equation: Y=Bmax [E2]total/(K_(d)+[E2]total). Theobserved binding affinities and extent of binding is compared to thedata from parallel binding studies carried out using an equivalentconcentration of non-immobilized ER-LBD. These studies will yieldbinding affinity/mg immobilized ER-LBD indices (BAI) which is used tocharacterize the immobilized receptor.

Optimization and reproducibility of the immobilization: Theimmobilization of the ERLBD is optimized through the investigation ofthe effect of ER-LBD concentration, reaction time, temperature andchemistry used in the immobilization. Each of the variables isindependently investigated in a step-wise optimization approach. Theoutcome of each iteration is assessed using the BAI. Once an optimumimmobilization procedure has been determined, the intra-day andinter-day reproducibility of the procedure is determined. A variance ofno greater than 10% is deemed acceptable. If this cannot be achievedunder the initially determined “optimal” conditions, other previouslydetermined conditions is investigated using the BIA as the selectingvariable.

Determination of the limits of quantitation and detection of theimmobilized ER-LBD chip: The estradiol ligand is derivatized withfluorescein-5-maleimide to produce the fluorescent-ligand {E2-FM} whichis used in the clinical patch. If this fluorescent-tag does not produceenough sensitivity, other agents are utilized. The immobilized ER-LBDchip is suspended over and then brought into surface contact withsolutions containing E2. The concentrations of the E2 solutions areserially diluted from the initial concentration of 0.050 nM untildisplacement of E2-FM can no longer be observed. The measured opticaldensity at λex=488 nm and λem=520 nm is plotted against the E2concentrations of the test solutions to construct standard curves.Standard inter-day and infra-day validation studies are conducted toestablish the reproducibility of the measurements, the lower limits ofquantitation and the lower limits of detection. Once this has beenestablished, the chip is ready for clinical testing.

Preparation of the AR-LBD: The androgen receptor ligand binding domain{AR-LBD} fusion protein is produced and purified following knownprocedures. Once the protein is expressed and purified, the bindingaffinity of the AR-LBD is determined by conventional methods followingthe procedure described for the ER-LBD.

Preparation of the immobilized AR-LBD chip and validation of itsactivity: The immobilization of the AR-LBD, determination of the bindingactivity of the immobilized ERLBD, determination of the limits ofquantitation and detection as well as the initial clinical validation iscarried out based upon the results obtained with the ER-LBD.

The protein is immobilized in a similar fashion as the ER-LBD, and theK_(d) is determined by frontal chromatography. The stability of thecolumn is also determined.

The immobilization of these receptors allows for rapid screening anddetermination of presence of biologically active/inactive compounds onthe estrogen and/or androgen receptors.

The recombinant ER-LBD is obtained and purified as described above.

Immobilization of the ER-LBD: The AR-LBD is then immobilized followingthe procedures described above for the ER-LBD.

The clinical trials described herein determine the analytical precisionand accuracy of methods to determine estrogen and testosterone viatransdermal sampling in comparison with validated plasma assays.Validated plasma ELISA assays for estrogen and testosterone withacceptable limits of detection and quantification and with acceptableintra- and inter-day coefficients of variation are used to compare withthese assays in the clinical settings described in the four trialsoutlined in detail below.

Clinical Trial 1: A Pilot Trial Correlating Estrogen Concentrations inplasma and Interstitial Fluid in Pre and Post-Menopausal Women. In orderto validate the analytical sensitivity of transdermal sampling tomeasure estrogen, eight healthy, menstruating women and eight healthypost-menopausal women who are taking no estrogen-containing medicationsare followed for two months to measure the estrogen concentrations intheir plasma or interstitial fluid. Concentrations of estrogen in theplasma is determined by a validated clinical ELISA assay, routinely usedin clinical settings. These concentrations are compared to thosedetermined in interstitial fluid using estrogen receptor-based assaysusing the HW monitoring device. Plasma and interstitial fluidconcentrations are measured daily during the period from the end ofafter menstruation until 10 days afterward in menstruating women andover a ten day period in post-menopausal women. Two groups of women arerecruited: a group of eight women who are pre-menopausal and who, byhistory, experience regular menstrual cycles; and a group of women whohave passed through menopause

Inclusion Criteria. Group 1: women who are over the age of 21 years andunder the age of 40 years. Group 2: women who are over the age of 55 andunder the age of 75. All women must conform with the following: (1)taking no prescription medications or natural products intended toproduce estrogen like effects (for example, ginseng or black kohosh);(2) with clinically normal laboratory values for complete blood counts,serum chemistries (Na, K, Cl, HCO₃, BUN, glucose and creatinine) andclinically normal liver enzyme profiles: SGOT, SGPT, alkalinephosphatase and bilirubin; (3) ability to understand and carry out asigned informed consent describing this protocol.

Exclusion Criteria. The following individuals are excluded from thestudy: (1)smokers; (2) impaired liver or renal function as demonstratedby serum SGOT, SGPT or bilirubin above the normal range of laboratoryvalues, or serum creatinine greater than 1.5 mg/dL; (3) positive urinedrug screen; (4) subjects who test positive for Human ImmunodeficiencyVirus or hepatitis; (5) subjects who have taken an investigational drugwithin 30 days of study start; (6) subjects taking any enzyme inducingor inhibiting medications (for example, rifampin or phenytoin) for 30days prior to dosing; and (7) subjects who, in the opinion of theInvestigator, is noncompliant with the protocol requirements.

Restrictions include that subjects are instructed to refrain from thefollowing: (1) taking any prescription medications for two weeks priorto dosing; (2) consuming caffeine and/or xanthine containing productsand alcohol from at least 48 hours prior to Study Day 1 until after thelast blood sample has been collected; (3) smoking; (4) strenuousexercise during the entire study to avoid dislocation of the measuringdevice.

Once a subject has consented to participate in the study, the followingprocedures are conducted. Screening procedures are conducted within 21days of study initiation and include: medical history and physicalexamination; review of inclusion and exclusion criteria; blood and urinespecimen collection; analysis of blood sample for hematology, serumchemistry, liver enzymes, HIV and hepatitis B and C; and analysis ofurine sample for screening of drugs of abuse.

Subsequent to inclusion in the study, subject will undergoes thefollowing procedures: subjects arrive at the testing facility atapproximately 9a.m. in the morning. While the precise date is notimportant for post-menopausal women, menstruating women are asked toreport on the last day of their regular period. At this time estrogenlevels are low and the detection limits of the assays is appropriatelytested. Vital signs (heart rate, respiratory rate, blood pressure andtemperature) are recorded. The portable biomedical monitoring device isplaced on a forearm and attached securely with tape. Recording occursvia a 50 micron cauterized lesion in the skin made by a small needle onthe underside of the monitor that is not visible. The monitor is checkedto ensure that it is recording. Vital signs (heart rate, respiratoryrate, blood pressure and temperature) are recorded.

A single sample of venous blood (5 cc or one teaspoon) is drawn from aforearm vein for the measurement of estrogen while the subject issupine. The device is instructed to measure estrogen for periods of0.25, 0.5, 1, 1.5, 2, 3, 6, and 8 hours in order to test the optimaltime required. Additional blood samples are drawn 4 hours and eighthours after the first. The tape and device is removed. Patients are thendischarged and allowed to return home before returning at 9 a.m. thenext morning. This procedure is repeated on the following 9 days for atotal of 10 days with each subject.

Blood Sampling Schedule. Five mL venous blood samples are collected invacutainers containing EDTA on days 1 through 10 in the manner describedabove. The total number of blood draws during the course of the studyincluding the screening and exit samples, is 35, (Thirty study draws andfive screening draws for hematology, chemistry, liver enzymes, HIV andHepatitis and C respectively.) and the total volume of blood drawn doesnot exceed 200 mL.

Vital signs (heart rate, respiratory rate, blood pressure andtemperature) are taken before and after placement of the device andbefore and after each blood draw on days 1 through 10.

Patients are encouraged to report any notable irritation on the armwhere the device is placed. A physician is constantly available tosubjects enrolled in the study for concerns related to bruising orinfection in the skin due to multiple blood draws.

Estrogen concentrations determined using plasma ELISA is compared withthe values obtained using the portable biomedical monitor. If thecorrelation coefficient is >0.8 with a significance p<0.05 themeasurements are deemed valid.

A Pilot Trial Correlating Testosterone Concentrations in plasma andInterstitial Fluid in Men. Ten healthy men, chosen to represent a rangeof ages between 21 and 70 years old and who are taking no medicationshave plasma and interstitial fluid testosterone concentrations measureddaily for 5 consecutive days both by validated plasma assay and bytransdermal sampling using the portable biomedical monitoring device.

Inclusion Criteria. Men who are over the age of 21 and under the age of75. All men must conform with the following: (1) taking no prescriptionmedications or natural products intended to produce testosterone likeeffects (for example, androstenedione or DHEA); (2) with clinicallynormal laboratory values for complete blood counts, serum chemistries(Na, K, Cl, HCO3, BUN, glucose and creatinine) and clinically normalliver enzyme profiles: SGOT, SGPT, alkaline phosphatase and bilirubin;and (3) ability to understand and carry out a signed informed consentdescribing this protocol.

Exclusion Criteria. The following individuals are excluded from thestudy: (1) smokers; (2) impaired liver or renal function as demonstratedby serum SGOT, SGPT or bilirubin above the normal range of laboratoryvalues, or serum creatinine greater than 1.5 mg/dL; (3) positive urinedrug screen; (4) subjects who test positive for Human ImmunodeficiencyVirus or hepatitis.

Restrictions include subjects are instructed to refrain from thefollowing: (1) smoking; and (2) strenuous exercise during the entirestudy to avoid dislocation of the measuring device.

Once a subject has consented to participate in the study, the followingprocedures are conducted: Screening procedures are conducted within 21days of study initiation and include: medical history and physicalexamination; review of inclusion and exclusion criteria; blood and urinespecimen collection; analysis of blood sample for hematology, serumchemistry, liver enzymes, HIV and hepatitis B and C; and analysis ofurine sample for screening of drugs of abuse.

Subsequent to inclusion in the study, subject will undergoes thefollowing procedures: Subjects arrive at the testing facility atapproximately 9 a.m. in the morning. Vital signs (heart rate,respiratory rate, blood pressure and temperature) are recorded. Theportable biomedical monitoring device is placed on a forearm andattached securely with tape. Recording occurs via a 50 micron cauterizedlesion in the skin made by a small needle on the underside of themonitor that is not visible. The monitor is checked to ensure that it isrecording. Vital signs (heart rate, respiratory rate, blood pressure andtemperature) are recorded.

A single sample of venous blood (5 cc or one teaspoon) is drawn from aforearm vein for the measurement of testosterone while the subject issupine. The device is instructed to measure estrogen for periods of0.25, 0.5, 1, 1.5, 2, 3, 6, and 8 hours in order to test the optimaltime required. Additional blood samples are drawn 4 hours and eighthours after the first. The tape and device is removed. Patients are thendischarged and allowed to return home before returning at 9 a.m. thenext morning. This procedure is repeated on the following 4 days for atotal of 5 days with each subject.

Five mL venous blood samples are collected in Vacutainers containingEDTA on Days 1 through 5 in the manner described above. The total numberof blood draws during the course of the study including the screeningand exit samples, is 20, (Fifteen study draws and five screening drawsfor hematology, chemistry, liver enzymes, HIV and Hepatitis and Crespectively.) and the total volume of blood drawn does not exceed 200mL.

Vital signs (heart rate, respiratory rate, blood pressure andtemperature) are taken before and after placement of the device andbefore and after each blood draw on days 1 through 5.

Patients are encouraged to report any notable irritation on the armwhere the device is placed. A physician is constantly available tosubjects enrolled in the study for concerns related to bruising orinfection in the skin due to multiple blood draws.

Testosterone concentrations determined using plasma ELISA is comparedwith the values obtained using the portable biomedical monitor. If thecorrelation coefficient is >0.8 with a significance p<0.05 themeasurements are deemed valid.

The purpose of this third clinical trial is to administer appropriateconcentrations of estrogen to postmenopausal women who are healthyvolunteers and to measure the resulting concentrations. Estrogen isadministered in microgram pulses over a period of 3 days after a 3-dayrun-in for baseline measurement using the optimal machine settings forthe monitor as defined above.

The purpose of this fourth clinical trial is to administer appropriateconcentrations of testosterone to men between the ages of 55 and 75years who are healthy volunteers and to measure the resultingconcentrations.

1-114. (canceled)
 115. A transdermal sampling system, comprising: atleast one sampler for retrieving and transferring at least one analyteobtained transdermally from the skin of a subject, wherein the sampleris a microfabricated device comprising a microfluidic assembly; at leastone detector system for identifying and quantifying said at least oneanalyte; and at least one logic module for (i) receiving and storinginput data from said at least one detector, (ii) relating the input datato other data obtained from the subject relating to the condition of thesubject, (iii) displaying output information indicative of health andclinical state of the subject as determined from the relating of theinput data to the other data, (iv) transmitting the output informationto another system, and (v) controlling the operation of said at leastone sampler and at least one detector.
 116. The system of claim 5,wherein said microfluidic assembly comprises: at least one reservoir; atleast one conduit in communication with the at least one reservoir,wherein the at least one conduit receives the at least one analyte fromthe at least one reservoir and transmits the at least one analytethrough the at least one conduit; and the at least one detector is inassociation with the at least one conduit to detect the at least oneanalyte transmitted through the at least one conduit.
 117. The system ofclaim 115, wherein said at least one sampler further comprises means forenhancing permeability of the skin of a subject for retrieving said atleast one analyte therefrom.
 118. The system of claim 117, wherein saidmeans for enhancing permeability comprises a microheater disposed to bein close proximity to a subject's skin surface, and configured forablating a portion of the stratum corneum of the skin of a subject andcollects of interstitial fluid from the subject's underlying viableepidermis.
 119. The system of claim 117, wherein said means forenhancing permeability comprises a microheater disposed to be in closeproximity to a subject's skin surface, and configured for ablating aportion of the stratum corneum of the skin of a subject and accessesinterstitial fluid from the subject's underlying viable epidermis. 120.The system of claim 117, wherein said means for enhancing permeabilityof the skin comprises a radiation source positioned for generating anddirecting radiation to a subject's skin surface, effective to ablate aportion of the stratum corneum of the skin of a subject, to allowcollection of interstitial fluid from the subject's underlying viableepidermis.
 121. The system of claim 120, wherein said radiation sourceis a light source.
 122. The system of claim 121, wherein said lightsource is a laser.
 123. The system of claim 117, wherein said means forenhancing permeability of the skin comprises a radiation sourcepositioned for generating and directing radiation to a subject's skinsurface, effective to ablate a portion of the stratum corneum of theskin of a subject, to allow diffusion from interstitial fluid from thesubject's underlying viable epidermis.
 124. The system of claim 117,wherein said means for enhancing permeability of the skin is at leastone of: (i) means for chemically altering the surface of the skin; (ii)means for puncturing the surface of the skin; (iii) means forsolubilizing the surface of the skin; (iv) means for illuminating thesurface of the skin sufficient to cause ablation thereof; and (v) meansfor irradiating the surface of the skin to cause ablation thereof. 125.The system of claim 115, wherein said at least one detector systemcomprises an optical detection system comprised of at least one lightsource effective to excite fluorophores, and at least one detector fordetecting fluorescence from excited fluorophores.
 126. The system ofclaim 125, wherein said light source comprises at least one LED or atleast one laser.
 127. The system of claim 116, wherein said at least onereservoir contains a fluid and transmits the fluid to a subject's skinand to the at least one conduit.
 128. The system of claim 127, whereinsaid fluid contained in said at least one reservoir permeates asubject's skin.
 129. The system of claim 127, wherein the reservoirretains the fluid in the reservoir with a breakable seal, wherein thebreaking of the breakable seal releases the fluid.
 130. The system ofclaim 115, further comprising an adhesive to substantially adhere thesampler to the skin of the subject.
 131. The system of claim 1 5,further comprising at least one substance located in said sampler whichis capable of binding an analyte of interest, and said substance beingdetectable by said at least one detector system.
 132. The system ofclaim 128, wherein a pump pumps the fluid into contact with the skin ofa subject.
 133. The system of claim 131, further comprising amicroheater disposed to be in close proximity to a subject's skinsurface at a location proximate to where said fluid is pumped intocontact with the skin of a subject, wherein the microheater ablates aportion of the stratum corneum of the skin of a subject.
 134. The systemof claim 115, further comprising means for monitoring predeterminedphysiological data as said other data, for relating to the input data bysaid at least one logic module.
 135. The system of claim 115, furthercomprising means for monitoring environmental conditions data as saidother data, for relating to the input data by said at least one logicmodule.
 136. The system of claim 115, wherein said at least one detectorsystem comprises a patch sensitive to at least one analyte for changingcolor in response to contact with said at least one analyte, and atleast one detector for detecting a change in color of the patch.
 137. Amicrofabricated device for allowing remote monitoring of a subject,comprising: at least one sampler unit body for retrieving andtransferring at least one analyte obtained transdermally from the skinof a subject, wherein the sampler unit body is a microfabricated devicecomprising a microfluidic assembly; at least one detector systemconnected to said at least one sampler unit body for identifying andquantifying at least one analyte obtained from a subject; and atransmitter/receiver for transmitting data relating to at least oneanalyte detected by said detection system to a logic module forprocessing thereby, and for allowing control of the microfabricateddevice by a logic module.
 138. The device of claim 137, wherein saidmicrofluidic assembly comprises: at least one reservoir; at least oneconduit in communication with the at least one reservoir, wherein the atleast one conduit receives the at least one analyte from the at leastone reservoir and transmits the at least one analyte through the atleast one conduit; and the at least one detector is in association withthe at least one conduit to detect the at least one analyte transmittedthrough the at least one conduit.
 139. The device of claim 137, whereinsaid at least one sampler unit body further comprises means forenhancing permeability of the skin of a subject for retrieving said atleast one analyte therefrom.
 140. The device of claim 139, wherein saidmeans for enhancing permeability comprises a microheater disposed to bein close proximity to a subject's skin surface, and configured forablating a portion of the stratum corneum of the skin of a subject toallow collection of interstitial fluid from the subject's underlyingviable epidermis.
 141. The device of claim 139, wherein said means forenhancing permeability of the skin comprises a radiation sourcepositioned for generating and directing radiation to a subject's skinsurface, effective to ablate a portion of the stratum corneum of theskin of a subject, to allow diffusion of interstitial fluid from thesubject's underlying viable epidermis.
 142. The device of claim 141,wherein said radiation source is a light source.
 143. The device ofclaim 142, wherein said light source is a laser.
 144. The device ofclaim 139, wherein said means for enhancing permeability of the skin isat least one of; (i) means for chemically altering the surface of theskin, (ii) means for puncturing the surface of the skin, (iii) means forsolubilizing the surface of the skin, (iv) means for illuminating thesurface of the skin sufficient to cause ablation thereof, and (v) meansfor irradiating the surface of the skin to cause ablation thereof. 145.The device of claim 137, wherein said at least one sampler unit bodycomprises a plurality of sampling conduits; and said at least onedetector system comprises a plurality of detector systems.
 146. Thedevice of claim 115, wherein said at least one detector system comprisesan optical detection system comprised of a light source effective toexcite fluorophores, and at least one detector for detectingfluorescence from excited fluorophores.
 147. The device of claim 146,wherein said light source is selected from the group consisting of atleast one LED or at least one laser.
 148. The device of claim 138,wherein said at least one reservoir contains a fluid and transmits thefluid to a subject's skin and to the at least one conduit.
 149. Thedevice of claim 148, wherein said fluid contained in said at least onereservoir permeates a subject's skin.
 150. The device of claim 148,wherein the reservoir retains the fluid in the reservoir with abreakable seal, wherein the breaking of the breakable seal releases thefluid.
 151. The device of claim 137, wherein the sampler unit bodysubstantially adheres to the skin of a subject.
 152. The device of claim137, further comprising at least one substance located in said samplerunit body which is capable of binding an analyte of interest, and saidsubstance being detectable by said at least one detector system. 153.The device of claim 137, wherein said at least one detector systemcomprises a patch sensitive to at least one analyte for changing colorin response to contact with said at least one analyte, positioned forcontact with said at least one analyte, and at least one detectorpositioned for detecting a change in color of the patch.
 154. The deviceof claim 137, wherein said sampler unit body is a silicon body having anarray of capillary conduits extending therethrough for sampling analytefrom the skin of a subject through said capillary conduits; and at leastone detector chamber associated with said at least one detection system,and in communication with said capillary conduits for detecting analytesreceived within the capillary chamber.
 155. The device of claim 154,wherein said sampler unit body further comprises means for enhancingpermeability of the skin of a subject for retrieving said at least oneanalyte therefrom.
 156. The device of claim 155, wherein said means forenhancing permeability comprises a microheater disposed to be in closeproximity to a subject's skin surface, and configured for ablating aportion of the stratum corneum of the skin of a subject to allowcollection of interstitial fluid from the subject's underlying viableepidermis.
 157. The device of claim 155, wherein said means forenhancing permeability comprises a microheater disposed to be in closeproximity to a subject's skin surface, and configured for ablating aportion of the stratum corneum of the skin of a subject to allowdiffusion from interstitial fluid from the subject's underlying viableepidermis.
 158. The device of claim 155, wherein said means forenhancing permeability of the skin comprises a radiation sourcepositioned for generating and directing radiation to a subject's skinsurface, effective to ablate a portion of the stratum corneum of theskin of a subject to allow collection of interstitial fluid from thesubject's underlying viable epidermis.
 159. The device of claim 155,wherein said means for enhancing permeability of the skin comprises aradiation source positioned for generating and directing radiation to asubject's skin surface, effective to ablate a portion of the stratumcorneum of the skin of a subject to allow diffusion of interstitialfluid from the subject's underlying viable epidermis.
 160. The device ofclaim 159, wherein said radiation source is a light source.
 161. Thedevice of claim 160, wherein said light source is a laser.
 162. Thedevice of claim 155, wherein said means for enhancing permeability ofthe skin is at least one of; (i) means for chemically altering thesurface of the skin, (ii) means for puncturing the surface of the skin,(iii) means for solubilizing the surface of the skin, (iv) means forilluminating the surface of the skin sufficient to cause ablationthereof, and (v) means for irradiating the surface of the skin to causeablation thereof.
 163. The device of claim 155, wherein said at leastone detector system comprises an optical detection system comprised oflight sources effective to excite fluorophores, and at least onedetector for detecting fluorescence from excited fluorophores.
 164. Thedevice of claim 163, wherein said light source comprises at least oneLED or at least one laser.
 165. The device of claim 155, wherein saidsilicon body comprises at least one reservoir in communication with saidcapillary conduits, wherein a fluid retained in the at least onereservoir transmits from the reservoir to the subject's skin into atleast one of the said capillary conduits.
 166. The device of claim 165,wherein said silicon body further comprises a microheater disposed to bein close proximity to a subject's skin surface at a location proximateto where said fluid is pumped into contact with the skin of a subject,wherein the microheater ablates a portion of the stratum corneum of theskin of a subject.
 167. A microfabricated device for sampling analytesfrom the skin of a subject, comprising: a detection chamber forreceiving analytes retrieved from the skin of a subject, wherein theanalytes are retrieved by a microfabricated device comprising amicrofluidic assembly; a photonic detection system, comprising aphotonics source located attached to said microfabricated device inassociation with said detection chamber; and at least one detectorassociated with said detection chamber for detecting analytes receivedin said detection chamber.
 168. The device of claim 167, wherein saidphotonics source is at least one LED or at least one laser.
 169. Thedevice of claim 167, further comprising substances in said device whichbind with selected analytes, and which fluoresce when said photonicssource applies radiation thereto.
 170. A microfabricated device forsampling analytes from the skin of a subject, comprising: a detectionchamber for receiving analytes retrieved from the skin of a subject,wherein the analytes are retrieved by a microfabricated devicecomprising a microfluidic assembly; a patch which changes color whencontacted by predetermined analytes, located attached to saidmicrofabricated device in association with said detection chamber; anddetectors associated with said detection chamber, for detecting a changeof color of the patch indicating the presence of a predeterminedanalyte.
 171. A device for sampling and detecting analytes retrievedfrom the skin of a subject, comprising: at least one microfluidicconduit for retrieving and transmitting an analyte from the skin of asubject to a detector, wherein the microfluidic conduit is amicrofabricated device; and means for enhancing permeability of the skinof a subject for retrieving said at least one analyte therefrom. 172.The device of claim 171, wherein said means for enhancing permeabilitycomprises a microheater disposed to be in close proximity to a subject'sskin surface, and configured for ablating a portion of the stratumcorneum of the skin of a subject to allow collection of interstitialfluid from the subject's underlying viable epidermis.
 173. The device ofclaim 171, wherein said means for enhancing permeability comprises amicroheater disposed to be in close proximity to a subject's skinsurface, and configured for ablating a portion of the stratum corneum ofthe skin of a subject to allow diffusion from interstitial fluid fromthe subject's underlying viable epidermis.
 174. The device of claim 171,wherein said means for enhancing permeability of the skin comprises aradiation source positioned for generating and directing radiation to asubject's skin surface, effective to ablate a portion of the stratumcorneum of the skin of a subject, to allow collection of interstitialfluid from the subject's underlying viable epidermis.
 175. The device ofclaim 171, wherein said means for enhancing permeability of the skincomprises a radiation source positioned for generating and directingradiation to a subject's skin surface, effective to ablate a portion ofthe stratum corneum of the skin of a subject, to allow diffusion frominterstitial fluid from the subject's underlying viable epidermis. 176.The device of claim 174, wherein said radiation source is a lightsource.
 177. The device of claim 176, wherein said light source is alaser.
 178. The device of claim 171, wherein said means for enhancingpermeability of the skin is at least one of: (i) means for chemicallyaltering the surface of the skin; (ii) means for puncturing the surfaceof the skin; (iii) means for solubilizing the surface of the skin; (iv)means for illuminating the surface of the skin sufficient to causeablation thereof; and (v) means for irradiating the surface of the skinto cause ablation thereof.
 179. The transdermal sampling systemaccording to claim 115, wherein the microfluidic assembly comprises atleast one serpentine capillary channel.
 180. The transdermal samplingsystem according to claim 115, wherein the microfluidic assembly furthercomprises: at least one reservoir channel: at least one bottom cappingsection: and at least one top capping section.
 181. The transdermalsampling system according to claim 180, wherein the at least onereservoir channel further comprises at least one seal for retaining aphysiologically compatible fluid within the at least one reservoirchannel.
 182. The transdermal sampling system according to claim 181,wherein the at least bottom capping section comprises a micro-heatingelement.
 183. The transdermal sampling system according to claim 182,wherein the micro-heating element serves to ablate stratum corneum. 184.The transdermal sampling system according to claim 182, wherein themicro-heating element serves to rupture the at least one seal.
 185. Thetransdermal sampling system according to claim 182, wherein themicro-heating element serves both to ablate stratum corneum and torupture the at least one seal.
 186. The transdermal sampling systemaccording to claim 180, wherein the at least one top capping sectioncomprises two or more electrodes.
 187. The transdermal sampling systemaccording to claim 186, wherein the two or more electrodes serve toassist the flow of a physiologically compatible fluid through an atleast one serpentine capillary channel.
 188. The transdermal samplingsystem according to claim 180, further comprising a sensor for detectingthe at least one analyte.
 189. The transdermal sampling system accordingto claim 188, wherein the sensor for detecting the at least one analytecomprises a patch sensitive to the at least one analyte for changingcolor in response to contact with the at least one analyte, and at leastone detector for detecting a change in color of the patch.
 190. Thetransdermal sampling system according to claim 115, wherein themicrofluidic assembly contains a physiologically compatible fluid thattransfers at least one analyte obtained transdermally from the skin of asubject.
 191. The transdermal sampling system according to claim 130,wherein the adhesive substantially prevents movement of the transdermalsampling system relative to the skin of the subject.
 192. Thetransdermal sampling system according to claim 130, wherein the adhesiveserves to prevent loss of the physiologically compatible fluid.
 193. Thetransdermal sampling system according to claim 130, wherein the adhesiveis water impermeable.
 194. The transdermal sampling system according toclaim 115, wherein at least one surface of the microfluidic assembly ismodified in a manner to enhance sampling function.
 195. The transdermalsampling system according to claim 194, wherein the modification of theat least one surface of the microfluidic assembly prevents theadsorption of protein to the at least one surface of the microfluidicassembly.
 196. The transdermal sampling system according to claim 194,wherein the modification of the at least one surface of the microfluidicassembly attaches to the at least one surface of the microfluidicassembly at least one specific-binding molecule which specifically bindsthe at least one analyte.
 197. A method of biomedically monitoring asubject's condition, the method comprising: ablating a subject's skin toallow interstitial fluid to perfuse therethrough; collectinginterstitial fluid from the subject's ablated skin, using amicrofabricated device comprising a microfluidic assembly; andidentifying and quantifying at least one type of selected moleculescontained in the collected interstitial fluid.
 198. The method of claim197, wherein the at least one type of molecule is a metabolic marker ofstress.
 199. The method of claim 197, wherein the at least one type ofmolecule is at least one of an organophosphate, microbial toxin,inflammatory sequeli to microbial toxin, spore metabolite, prealbumin,C-reactive protein, troponin I, estrogen, and testosterone.
 200. Themethod of claim 197, further comprising monitoring at least one of thesubject's vital physiologically statistics.
 201. The method of claim200, wherein said at least one of the subject's vital physiologicalstatistics comprises at least one of body temperature, pulse rate, bloodpressure, and heart activity.
 202. The method of claim 197, furthercomprising monitoring and detecting environmental conditions in whichthe subject is located.
 203. The method of claim 197, wherein saididentifying and quantifying comprises transferring said at least onetype of molecule to a patch which changes color when in contact withsaid at least one type of molecule, and detecting a change in color ofthe patch.
 204. The method of claim 197, wherein said identifying andquantifying comprises binding the at least one type of molecule with asecond type of molecule which fluoresces when irradiated, irradiatingthe at least one type of molecule bound to the second type of molecule,and detecting any resultant fluorescence.
 205. The method of claim 204,wherein the source for irradiating is a laser or an LED.
 206. Thetransdermal sampling system according to claim 129, wherein the seal issemi-permeable or selectively permeable.
 207. The transdermal samplingsystem according to claim 150, wherein the seal is semi-permeable orselectively permeable.
 208. The transdermal sampling system according toclaim 181, wherein the seal is semi-permeable or selectively permeable.